Gene family with transformation modulating activity

ABSTRACT

pp32 is a member of a highly conserved family of differentiation-regulated nuclear proteins that is highly expressed in nearly all human prostatic adenocarcinomas of Gleason Grade ≧5. This contrasts with the low percentage of prostate tumors that express molecular alterations in proto-oncogens or demonstrate tumor suppressor mutation or loss of heterozygosity. By analysis of specimens of human prostatic adenocarcinoma and paired adjacent normal prostate from three individual patients, the inventors have shown that normal prostate continues to express normal pp32, whereas three of three sets of RT-PCR-amplified transcripts from prostatic adenocarcinomas display multiple cancer-associated coding sequence changes. The cancer-associated sequence changes appear to be functionally significant. Normal pp32 exerts antineoplastic effects through suppression of transformation. In contrast, cancer-associated pp32 variants augment, rather than inhibit, transformation.

[0001] The work leading to this invention was supported in part by GrantNo. RO1 CA 54404 from the National Institutes of Health. The U.S.Government retains certain rights in this invention.

BACKGROUND

[0002] 1. File of the Invention

[0003] This invention is directed to various members of a gene familywith transformation modulating activity, and to diagnostic and genetherapy techniques based on the variants.

[0004] 2. Review of Related Art

[0005] Prostatic adenocarcinoma is the most frequent malignancy in adultmen with approximately 317,000 new cases diagnosed each year (Parker, etal., CA, 46:8-27, 1996). In spite of the capabilities for earlydiagnosis and treatment (Potosky, et al., JAMA, 273:548-552, 1995), itrepresents the second leading cause of cancer death in men followinglung cancer.

[0006] To date, the study of alterations in specific genes has not beenparticularly rewarding in primary prostate cancer. Most alterations inthe widely studied oncogenes and tumor suppressor genes occur in only20-30% of primary prostate carcinomas, except for the myc gene, whereoverexpression has been observed in as many as 50-60% of such cases(Fleming, et al., Cancer Res., 46:1535-1538, 1986). Up to 40% of primaryprostate cancers studied by comparative genomic hybridization displaychromosomal aberrations (Visakorpi, et al., Cancer Res., 55:342-347,1995), although such alterations occur more frequently as tumors recurand become refractory to hormonal therapy. Characterization of candidateproto-oncogenes or tumor suppressor genes at such altered loci mayeventually shed light on tumor progression in the prostate.

[0007] pp32 (GenBank HSU73477) is a highly conserved nuclearphosphoprotein. Increased expression of pp32 or closely related speciesis a frequent feature of clinical cancers. For example, in humanprostate cancer, high-level expression of RNA hybridizing with pp32probes occurs in nearly 90% of clinically significant prostate cancers,in contrast to the substantially lower frequencies of alterations ofother oncogenes and tumor suppressors (See U.S. Pat. No. 5,726,018,incorporated herein by reference). Molecular Features and Activities ofpp32.

[0008] pp32 is a nuclear phosphoprotein that isdifferentiation-regulated during differentiation of adult prostaticepithelium (Walensky, et al., Cancer Res. 53:4720-4726, 1993). The humanpp32 cDNA sequence (Gen-Bank U73477) is 1052 bp in length and encodes aprotein of 249 amino acids. The protein is composed of two domains: anamino terminal amphipathic α-helical region containing a leucine zipper,and a highly acidic carboxyl terminal region. The murine and human formsof pp32 are highly conserved with over 90% nucleic acid homology andover 95% protein-level homology.

[0009] Human pp32 has been isolated independently by a number of groups.Vaesen et al. (“Purification and characterization of two putative HLAclass II associated proteins: PHAPI and PHAPII.” Biol. Chem.Hoppe-Seyler., 375:113-126, 1994) cloned an essentially equivalentmolecule, termed PHAPI, from an EBV-transformed human B-lymphoblastoidcell line; PHAPII, cloned by the same strategy, is unrelated to pp32.This study identified PHAPI through its association in solution withhuman HLA class II protein, noting membrane and cytoplasmic localizationas well as nuclear; the gene has putatively been localized to chromosome15q22.3-q23 by fluorescent in situ hybridization (Fink, et al.,“Localization of the gene encoding the putative human HLA classII-associated protein (PHAPI) to chromosome 15q22.3-q23 by fluorescencein situ hybridization.” Genomics, 29:309-310, 1995). More recently, agroup studying inhibitors of protein phosphatases identified pp32 asIIPP2a, an inhibitor of protein phosphatase 2a (Li, et al., “MolecularIdentification of II PP2A, a novel potent-heat-stable inhibitor proteinof protein phosphatase 2A.” Biochemistry 35:6998-7002, 1996); anotherphosphatase inhibitor, I2PP2a, is unrelated to pp32. Interestingly,another recent report (Ulitzur, et al., “Biochemical characterization ofmapmodulin, a protein that binds microtubule-associated proteins.”Journal of Biological Chemistry 272:30577-30582, 1997) identified pp32as a cytoskeletally-associated cytosolic protein in CHO cells. It is notclear whether this finding stems from a difference in system, or whetherpp32 can localize to the cytoplasm under certain circumstances. pp32 hasalso been identified as LANP, a leucine rich nuclear protein in thecentral nervous system (Matsuoka, et al., “A nuclear factor containingthe leucine-rich repeats expressed in murine cerebellar neurons. ProcNatl Acad Sci USA 91:9670-9674, 1994).

[0010] There are also a number of reports of gene products bearinglesser degrees of homology to pp32. The Vaesen group has identified aseries of unpublished sequences, termed PHAPI2a (EMBL Locus HSPHAPI2A)and PHAPI2b (EMBL Locus HSPHAPI2B), also cloned from an EBV-transformedhuman B-lymphoblastoid cell line. These variant pp32 sequences aredistinct from the sequences reported herein, representing the Aprilprotein instead. April, cloned from human pancreas, is shorter thanPHAPI2a by two N-terminal amino acids (Mencinger, et al., “Expressionanalysis and chromosomal mapping of a novel human gene, APRIL, encodingan acidic protein rich in leucines.” Biochimica et Biophysica Acta,1395:176-180, 1998, see EMBL Locus HSAPRIL); PHAPI2b is identical to asubset of APRIL. Silver-stainable protein SSP29 (unpublished GenBankLocus HSU70439) was cloned from HeLa cells and is identical to PHAPI2a.

[0011] The nuclear phosphoprotein pp32 has been linked to proliferation.Malek and associates reported that various neoplastic cell lines showedmarkedly elevated expression levels and that bacterial polysaccharideinduced expression of pp32 epitopes by B lymphocytes upon polyclonalexpansion (Malek, et al., J. Biol. Chem., 265:13400-13409, 1990).Walensky and associates reported that levels of pp32 expression,measured by in situ hybridization, increased in direct relation toincreasing Gleason grade of human prostatic cancers.

[0012] pp32 cDNA probes hybridize strongly with prostaticadenocarcinoma, whereas the hybridization signal in normal prostate isconfined to basal cells. Polyclonal anti-pp32 antibodies react stronglywith sections of human prostatic adenocarcinoma. The antibodies andriboprobes used by the investigators in previous studies are consistentwith cross-reactivities of the reagents with all reported members of thepp32 nuclear phosphoprotein family, therefore, while previousdescriptions focused upon pp32, it cannot be excluded that homologousproteins were detected.

SUMMARY OF THE INVENTION

[0013] In one aspect, this invention provides a DNA molecule containingat least a portion of the sequence consisting of base pairs 4894-4942 ofthe sequence shown in FIG. 2 or its complement. Alternatively, the DNAmolecule may contain at least a portion consisting of base pairs4879-4927, or base pairs 4858-4927. Alternatively, this inventionprovides a DNA molecule that contains at least a portion of a nucleotidesequence encoding amino acid residues 146-163 of tumor-derived pp32r1sequence; preferably the DNA encodes all of that segment. In one mode,the DNA molecule is an expression vector which expresses said amino acidsequence, and the invention also includes a recombinant cell containingthe expression vector. In another mode, the DNA molecule has theparticular sequence operatively linked to a promoter in antisenseorientation. In another alternative, this invention provides a DNA probewhich specifically hybridizes on Northern blot with nucleic acidencoding the amino acids from residue 146-163 of the tumor-derivedpp32r1 sequence, a preferred probe would have a sequence of at least 8contiguous nucleotides “unique” to the nucleotide sequence of the pp32r1variant as described herein. In yet another alternative, the inventionprovides a pair of nucleic acid primers each of which comprises at least10 contiguous nucleotides, at least one of the primers bindingspecifically to the pp32r1 sequence, where if the primers are used innucleic acid amplification of a suitable source of human nucleic acid,the amplification will produce an amplified nucleic acid encoding atleast residues 146-163 of the pp32r1 sequence.

[0014] In still another aspect, this invention provides antibodies thatspecifically bind the tumor derived pp32, but do not bind to normalpp32. Preferably, these antibodies are monoclonal antibodies. Theinvention also provides polypeptides containing epitopes that bind theseantibodies.

[0015] In yet another aspect, this invention provides diagnostic methodsfor predicting malignant potential of neuroendocrine, neural,mesenchymal, lymphoid, epithelial or germ cell derived tumors bydetermining, in a sample of human neuroendocrine, neural, mesenchymal,lymphoid, epithelial or germ cell derived tissue, the level of, or theintracellular sites of expression of, a gene product expressed from agene sequence which encodes, inter alia, residues 146-163 of tumorderived pp32r1. Where the gene product is mRNA, the mRNA is extractedfrom the sample and quantitated, optionally by PCR, or the level of mRNAmay be determined by in situ hybridization to a section of the tissuesample. Where the gene product is protein, the determination may includereacting the sample with an antibody that specifically binds to tumorderived pp32, but not to normal pp32. Preferably, the tissue sample iscarcinoma tissue. e.g., carcinoma or sarcoma of a tissue selected fromthe group consisting of epithelial, lymphoid, hematopoietic,mesenchymal, central nervous system and peripheral nervous systemtissues, including colon carcinoma, prostate carcinoma and non-Hodgkin'slymphoma.

[0016] In still another aspect, this invention provides anandrogen-activated transcriptional promoter which may be inserted intorecombinant DNA molecules. The minimal promoter is made up of atranscription initiation site and at least one binding site for asteroid hormone receptor protein. Typically the consensus sequence forthe steroid hormone receptor protein binding site is positioned within5000 nucleotide base pairs (bp), more preferably within 3000 bp, or evenfewer bp of the transcription initiation site: In a preferred mode, anumber of binding sites for steroid hormone receptor proteins arepositioned within that distance of the transcription initiation site,the promoter may contain five, ten or even 25 steroid hormone receptorprotein binding sites. Preferably, the binding site(s) for steroidhormone receptor protein binding are selected from the consensussequences listed on Table 1. In a preferred mode of the invention, theandrogen-activated transcriptional promoter is operatively linked to anopen reading frame comprising at least one exon of a protein codingsequence, operative linking of the open reading frame thereby providingan expression vector in which expression of the open reading frame isregulated by steroids.

[0017] In another aspect, this invention provides a method for screeningcandidate compounds for pharmacological activity by (1) culturing a celltransfected with the DNA molecule containing the androgen-activatedtranscriptional promoter which is operatively linked to an open readingframe comprising at least one exon of a protein coding sequence, and (2)determining expression of the open reading frame in the presence andabsence of the compound. In a preferred mode the androgen-activatedpromoter may be all or an operative portion of the sequence in FIG. 2which is up-stream of the translation initiation site, or alternativelythe androgen-activated promoter may be the 2700 bp of the sequence inFIG. 2 which is upstream from the translation initiation site.

[0018] pp32 is a member of a highly conserved family ofdifferentiation-regulated nuclear proteins that is highly expressed innearly all human prostatic adenocarcinomas of Gleason Grade ≧5. Thiscontrasts with the low percentage of prostate tumors that expressmolecular alterations in proto-oncogenes or demonstrate tumor suppressormutation or loss of heterozygosity. By analysis of specimens of humanprostatic adenocarcinoma and paired adjacent normal prostate from threeindividual patients, the inventors have shown that normal prostatecontinues to express normal pp32, whereas three of three sets ofRT-PCR-amplified transcripts from prostatic adenocarcinomas displaymultiple cancer-associated coding sequence changes. Thecancer-associated sequence changes appear to be functionallysignificant. Normal pp32 exerts antineoplastic effects throughsuppression of transformation. In contrast, cancer-associated pp32variants augment, rather than inhibit, transformation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1A shows detection of pp32-related mRNA in benign prostateand prostate cancer tissue sections by in situ hybridization.

[0020]FIG. 1B shows immunohistochemical stain of prostate cancersections with anti-pp32 antibodies.

[0021]FIG. 2 shows the genomic sequence of variant pp32r1 isolated fromhuman placenta.

[0022]FIG. 3 provides a base-by-base comparison of the sequence ofpp32r1 (top) with normal human pp32 (bottom). The numbering system forpp32r1 corresponds to FIG. 1, and the numbering system for normal pp32is taken from Chen, et al. Nucleotide base differences are underlined inthe pp32r1 sequence. Sequences within the normal pp32 sequence missingin pp32r1 are represented by dashes. The open reading frame for pp32r1is indicated by overlining.

[0023]FIG. 4 shows the alignment of the pp32r1 amino acid sequence (top)with normal human pp32 (bottom). Residue changes are underlined in thepp32r1 sequence. Amino acids missing in the pp32r1 sequence compared tonormal pp32 are represented by dashes.

[0024]FIG. 5 shows the genomic sequence of variant pp32r2.

[0025]FIG. 6A shows RT-PCR amplification of pp32 and pp32 variants fromhuman prostate cancer and prostate cancer cell line.

[0026]FIG. 6B shows cleavase fragment length polymorphism analysis ofpp32 detects variant pp32 transcripts in human prostate cancer.

[0027]FIG. 7 shows the alignment of nucleic acid (A) and amino acid (B)sequences from human prostatic adenocarcinoma and prostaticadenocarcinoma cell lines with pp32.

[0028]FIG. 8 is a bar graph showing ras+myc induced transformed focusformation. Co-transfection with a pp32 expression vector reducestransformation, while co-transfection with a pp32r1 expression vectorstimulates transformation.

[0029]FIG. 9 is a bar graph showing pp32r1 stimulation of ras+mycinduced transformed focus formation. Co-transfection with a pp32expression vector reduces transformation, while co-transfection withexpression vectors for pp32r1 sequences from prostate cancer cell linesstimulate transformation.

[0030]FIG. 10 is a graph of transformation assay results for cellstransfected with variant pp32 species, which are shown to stimulatetransformation with variable potency.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The inventors have discovered that phenotypic changes in pp32 area common feature of human prostate cancer. Previous data show that 87%of prostate cancers of Gleason Score 5 and above express pp32 orclosely-related transcripts (U.S. Pat. No. 5,734,022, incorporatedherein by reference). This is striking in comparison to the frequency ofmolecular alterations in other widely studied oncogenes and tumorsuppressor genes in primary prostatic adenocarcinoma, which occur in asubstantially smaller proportion of cases. For example, mycoverexpression (Fleming, et al.) occurs in around 60% of cases, and p53is abnormal in only around 25% of primary tumors (Isaacs, et al., in“Genetic Alterations in Prostate Cancer.” Cold Spring Harbor Symposia onQuantitative Biology, 59:653-659, 1994).

[0032] Several lines of evidence suggest that pp32 may act as a tumorsuppressor. Functionally, pp32 inhibits transformation in vitro byoncogene pairs such as ras with myc, mutant p53, Ela, or jun, or humanpapilloma virus E6 and E7 (Chen, et al., “Structure of pp32, an acidicnuclear protein which inhibits oncogene-induced formation of transformedfoci.” Molecular Biology of the Cell, 7:2045-2056, 1996). pp32 alsoinhibits growth of transformed cells in soft agar (Chen, et al.). Inanother system, ras-transfected NIH3T3 cells previously transfected tooverexpress normal human pp32 do not form foci in vitro or,preliminarily, do not form tumors in nude mice, unlike control cells. Incontrast, knockout of endogenous pp32 in the same system by an antisensepp32 expression construct markedly augments tumorigenesis (Example 12below).

[0033] In clinical prostate cancer, the situation at first appearscounterintuitive. Most human prostate cancers seem to express highlevels of pp32 by in situ hybridization (see Example 1 below) and stainintensely with anti-pp32 antibodies. Because pp32 inhibitsoncogene-mediated transformation (Chen, et al.), its paradoxicalexpression in cancer was investigated at the sequence level. Theparadoxical question of why prostate cancers seem to express high-levelsof an anti-oncogenic protein was addressed by comparing the sequence andfunction of pp32 species from paired normal prostate and adjacentprostatic carcinoma from three patients as well as from four prostatecancer cell lines. It is demonstrated herein that pp32 is a member of aclosely-related gene family, and that alternate expression of theseclosely-related genes located on different chromosomes modulatesoncogenic potential in human prostate cancer. The variant pp32 speciesexpressed in prostate cancer are closely related to pp32.

[0034] The present data indicate that prostate cancers express variantpp32 transcripts, whereas adjacent normal prostate expresses normalpp32. Two instances clearly show that expression of alternate genes ondifferent chromosomes can lead to the phenotypic switch, rather thanmutation or alternate splicing. This switch in molecular phenotype isaccompanied by a switch in functional pp32 phenotype. Normal pp32 isanti-oncogenic in character, in contrast to the pro-oncogenic varianttranscripts that foster oncogene-mediated transformation. The highfrequency of this abnormality suggests that expression of variant pp32species may play an etiologic role in the development of human prostatecancer. In addition, these findings have significant diagnostic andprognostic implications.

[0035] Definitions

[0036] In describing the present invention, the following terminology isused in accordance with the definitions set out below.

[0037] Nucleic Acids

[0038] In discussing the structure of particular double-stranded DNAmolecules, sequences may be described herein according to the normalconvention of giving only the sequence in the 5′ to 3′ direction alongthe nontranscribed stand of DNA (i.e., the-strand having a sequencehomologous to the mRNA).

[0039] A DNA sequence “corresponds” to an amino acid sequence iftranslation of the DNA sequence in accordance with the genetic codeyields the amino acid sequence (i.e., the DNA sequence “encodes” theamino acid sequence): one DNA sequence “corresponds” to another DNAsequence if the two sequences encode the same amino acid sequence.

[0040] Two DNA sequences are “substantially similar” when at least about90% (preferably at least about 94%, and most preferably at least about96%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially similar can be identified bythe assay procedures described below or by isolating and sequencing theDNA molecules. See e.g., Maniatis et al., infra, DNA Cloning, vols. 1and II infra: Nucleic Acid Hybridization, infra.

[0041] A “heterologous” region or domain of a DNA construct is anidentifiable segment of DNA within a larger DNA molecule that is notfound in association with the larger molecule in nature. Thus, when theheterologous region encodes a mammalian gene, the gene will usually beflanked by DNA that does not flank the mammalian genomic DNA in thegenome of the source organism. Another example of a heterologous regionis a construct where the coding sequence itself is not found in nature(e.g., a cDNA where the genomic coding sequence contains introns, orsynthetic sequences having codons different than the native gene).Allelic variations or naturally-occurring mutational events do not giverise to a heterologous region of DNA as defined herein.

[0042] A “coding sequence” or “open reading frame” is an in-framesequence of codons that (in view of the genetic code) correspond to orencode a protein or peptide sequence. Two coding sequences correspond toeach other if the sequences or their complementary sequences encode thesame amino acid sequences. A coding sequence in association withappropriate regulatory sequences may be transcribed and translated intoa polypeptide in vivo. A polydenylation signal and transcriptiontermination sequence will usually be located 3′ to the coding sequence.A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. Promoter sequences typically containadditional sites for binding of regulatory molecules (e.g.,transcription factors) which affect the transcription of the codingsequence. A coding sequence is “under the control” of the promotersequence or “operatively linked” to the promoter when RNA polymerasebinds the promoter sequence in a cell and transcribes the codingsequence into mRNA, which is then in turn translated into the proteinencoded by the coding sequence.

[0043] Vectors are used to introduce a foreign substance, such as DNA,RNA or protein, into an organism. Typical vectors include recombinantviruses (for DNA) and liposomes (for protein). A “DNA vector” is areplicon, such as plasmid, phase or cosmid, to which another DNA segmentmay be attached so as to bring about the replication of the attachedsegment. An “expression vector” is a DNA vector which containsregulatory sequences which will direct protein synthesis by anappropriate host cell. This usually means a promoter to bind RNApolymerase and initiate transcription of mRNA, as well as ribosomebinding sites and initiation signals to direct translation of the mRNAinto a polypeptide. Incorporation of a DNA sequence into an expressionvector at the proper site and in correct reading frame, followed bytransformation of an appropriate host cell by the vector, enables theproduction of a protein encoded by said DNA sequence.

[0044] An expression vector may alternatively contain an antisensesequence, where a small DNA fragment, corresponding to all or part of anmRNA sequence, is inserted in opposite orientation into the vector aftera promoter. As a result, the inserted DNA will be transcribed to producean RNA which is complementary to and capable of binding or hybridizingwith the mRNA. Upon binding to the mRNA, translation of the mRNA isprevented, and consequently the protein coded for by the mRNA is notproduced. Production and use of antisense expression vectors isdescribed in more detail in U.S. Pat. No. 5,107,065 (describing andexemplifying antisense regulation of genes in plants) and U.S. Pat. No.5,190,931 (describing antisense regulation of genes in both prokaryotesand eukarvotes and exemplifying prokaryotes), both of which areincorporated herein by reference.

[0045] “Amplification” of nucleic acid sequences is the in vitroproduction of multiple copies of a particular nucleic acid sequence. Theamplified sequence is usually in the form of DNA. A variety oftechniques for carrying out such amplification are described in a reviewarticle by Van Brunt (1990, Bio/Technol., 8(4):291-294). Polymerasechain reaction or PCR is a prototype of nucleic acid amplification anduse of PCR herein should be considered exemplary of other suitableamplification techniques.

[0046] Polypeptides

[0047] For the purposes of defining the present invention, two proteinsare homologous if 80% of the amino acids in their respective amino acidsequences are the same; for proteins of differing length, the sequenceswill be at least 80% identical over the sequence which is in common(i.e., the length of the shorter protein).

[0048] Two amino acid sequences are “substantially similar” when atleast about 87% of the amino acids match over the defined length of theamino acid sequences, preferably a match of at least about 89%, morepreferably a match of at least about 95%. Typically, two amino acidsequences which are similar will differ by only conservativesubstitutions.

[0049] “Conservative amino acid substitutions” are the substitution ofone amino acid residue in a sequence by another residue of similarproperties, such that the secondary and tertiary structure of theresultant peptides are substantially the same. Conservative amino acidsubstitutions occur when an amino acid has substantially the same chargeor hydrophobicity as the amino acid for which it is substituted and thesubstitution has no significant effect on the local conformation of theprotein. Amino acid pairs which may be conservatively substituted forone another are well-known to those of ordinary skill in the art.

[0050] The polypeptides of this invention encompass pp32r1 and pp32r1analogs, pp32r2 and pp32r2 analogs, along with other variants of pp32and their analogs. pp32r1 and pp32r2 are naturally occurring, matureproteins, and further encompass all precursors and allelic variations ofpp32r1 and pp32r2, as well as including forms of heterogeneous molecularweight that may result from inconsistent processing in vivo. An exampleof the pp32r1 sequence is shown in FIG. 3, top line. “pp32r1 analogs”are a class of peptides which includes:

[0051] 1) “Allelic variations of pp32r1,” which are polypeptides whichare substantially similar to pp32r1. Preferably the amino acid sequenceof the allelic variation is encoded by a nucleic acid sequence thatdiffers from the sequence of pp32r1 by one nucleotide in 300;

[0052]2) “Truncated pp32r1 peptides,” which include fragments of eitherpp32 or allelic variations of pp32r1 that preferably retain either (i)an amino acid sequence unique to pp32r1, (ii) an epitope unique topp32r1 or (iii) pp32r1 activity;

[0053] 3) “pp32r1 fusion proteins,” which include heterologouspolypeptides which are made up of one of the above polypeptides (pp32r1,allelic variations of pp32r1 or truncated pp32r1 peptides) fused to anyheterologous amino acid sequence.

[0054] “Unique” sequences of the pp32r1 variant according to thisinvention, either amino acid sequences or nucleic acid sequences whichencode them, are sequences which are identical to a sequence of a pp32r1polypeptide, but which differ in at least one amino acid or nucleotideresidue from the sequences of human pp32 (Genbank Locus HSU73477),murine pp32 (Genbank Locus MMU73478), human cerebellar leucine richacidic nuclear protein (LANP) (Genbank Locus AF025684), murine LANP(Genbank Locus AF022957). IIPP2a or human potent heat-stable proteinphospatase 2a inhibitor (Genbank Locus HSU60823), SSP29 (Genbank LocusHSU70439), HLA-DR associated protein 1 (Genbank Locus HSPPHAPI,Accession No. X75090), PHAPI2a (EMBL Locus HSPHAPI2A, Genbank AccessionNo. Y07569), PHAPI2b (EMBL Locus HSPHAPI2B, Genbank Accession No.Y07570), and April (EMBL Locus HSAPRIL), and preferably, are not foundelsewhere in the human genome. (A list of these sequences is provided inTable 3A.) Similarly, an epitope is “unique” to pp32r1 polypeptides ifit is found on pp32r1 polypeptides but not found on any members of theset of proteins listed above. Analogs of pp32r2 and unique pp32r2sequences are defined similarly. Of course, unique sequences of pp32r1are not found in pp32r2 and vice versa.

[0055] “Variants of pp32” are homologous proteins which differ from pp32by at least 2 amino acids. In particular, sequence comparison betweenpp32 and a variant will demonstrate at least one segment of 10 aminoacids in which the sequence differs by at least two (2) amino acids.More typically a variant will exhibit at least two such 10 amino acidsegments. Preferably, variants of pp32 in accordance with this inventionwill exhibit differences in functional activity from pp32. Inparticular, pp32r1 and pp32r2 are variants of pp32 whose activityincludes stimulation of transformation in the rat fibroblasttransformation assay described herein.

[0056] A composition comprising a selected component A is “substantiallyfree” of another component B when component A makes up at least about75% by weight of the combined weight of components A and B. Preferably,selected component A comprises at least about 90% by weight of thecombined weight, most preferably at least about 99% by weight of thecombined weight. In the case of a composition comprising a selectedbiologically active protein, which is substantially free ofcontaminating proteins, it is sometimes preferred that the compositionhaving the activity of the protein of interest contain species with onlya single molecular weight (i.e.. a “homogeneous” composition).

[0057] As used herein, a “biological sample” refers to a sample oftissue or fluid isolated from a individual, including but not limitedto, for example, plasma, serum, spinal fluid, lymph fluid, the externalsections of the skin, respiratory, intestinal, and genitourinary tracts,tears, saliva, milk, blood cells, tumors, organs, and also samples of invivo cell culture constituents (including but not limited to conditionedmedium resulting from the growth of cells in cell culture medium,putatively virally infected cells, recombinant cells, and cellcomponents).

[0058] “Human tissue” is an aggregate of human cells which mayconstitute a solid mass. This term also encompasses a suspension ofhuman cells, such as blood cells, or a human cell line.

[0059] The term “immunoglobulin molecule” encompasses whole antibodiesmade up of four immunoglobulin peptide chains, two heavy chains and twolight chains, as well as immunoglobulin fragments. “Immunoglobulinfragments” are protein molecules related to antibodies, which are knownto retain the epitopic binding specificity of the original antibody suchas Fab, F(ab)′₂, Fv, etc. Two polypeptides are “immunologicallycross-reactive” when both polypeptides react with the same polyclonalantiserum.

[0060] General Methods

[0061] The practice of the present invention employs, unless otherwiseindicated, conventional molecular biology, microbiology, and recombinantDNA techniques within the skill of the art. Such techniques are wellknown to the skilled worker and are explained fully in the literature.See, e.g., Maniatis, Fritsch & Sambrook, “Molecular Cloning: ALaboratory Manual” (1982); “DNA Cloning: A Practical Approach,” VolumesI and II (D. N. Glover, ed., 1985); “Oligonucleotide Synthesis” (M. J.Gait. ed., 1984); “Nucleic Acid Hybridization” (B. D. Hames & S. J.Higgins, eds., 1985): “Transcription and Translation” (B. D. Hames & S.J. Higgins, eds., 1984): “Animal Cell Culture” (R. I. Freshney, ed.,1986); “Immobilized Cells and Enzymes” (IRL Press, 1986); B. Perbal, “APractical Guide to Molecular Cloning” (1984), and Sambrook, et al.,“Molecular Cloning: a Laboratory Manual” (1989).

[0062] pp32 Related Genomic DNA

[0063] Screening a human genomic library in bacteriophages with probesgenerated from human pp32 cDNA yielded a new sequence that contained anopen reading frame encoding a protein homologous with pp32 (see Example2; pp32 sequence, reported in Chen, et al., Mol. Biol. Cell,7:2045-2056, 1996). While the pp32r1 and pp32r2 sequences (see FIGS. 2and 5) are substantially homologous to pp32, multiple single nucleotidebase changes and short deletions suggest that they are encoded by genedistinct from pp32 gene. The pp32 family also includes substantiallyhomologous polypeptides reported by others: HLA-DR associated protein 1(Vaesen, 1994), leucine-rich acidic nuclear protein (Matsuoka, 1994),and protein phosphatase 2A inhibitor (Li, 1996).

[0064] DNA segments or oligonucleotides having specific sequences can besynthesized chemically or isolated by one of several approaches. Thebasic strategies for identifying, amplifying and isolating desired DNAsequences as well as assembling them into larger DNA moleculescontaining the desired sequence domains in the desired order, are wellknown to those of ordinary skill in the art. See. e.g., Sambrook, etal., (1989); B. Perbal, (1984). Preferably, DNA segments correspondingto all or a part of the cDNA or genomic sequence of pp32r1 may beisolated individually using the polymerase chain reaction (M. A. Innis,et al., “PCR Protocols: A Guide To Methods and Applications.” AcademicPress, 1990). A complete sequence may be assembled from overlappingoligonucleotides prepared by standard methods and assembled into acomplete coding sequence. See, e.g., Edge (1981) Nature 292:756:Nambair, et al. (1984) Science 223:1299: Jay, et al. (1984) J. Biol.Chem., 259:6311.

[0065] The assembled sequence can be cloned into any suitable vector orreplicon and maintained there in a composition which is substantiallyfree of vectors that do not contain the assembled sequence. Thisprovides a reservoir of the assembled sequence, and segments or theentire sequence can be extracted from the reservoir by excising from DNAin the reservoir material with restriction enzymes or by PCRamplification. Numerous cloning vectors are known to those of skill inthe art, and the selection of an appropriate cloning vector is a matterof choice (see, e.g., Sambrook, et al., incorporated herein byreference). The construction of vectors containing desired DNA segmentslinked by appropriate DNA sequences is accomplished by techniquessimilar to those used to construct the segments. These vectors may beconstructed to contain additional DNA segments, such as bacterialorigins of replication to make shuttle vectors (for shuttling betweenprokaryotic hosts and mammalian hosts), etc.

[0066] Procedures for construction and expression of proteins of definedsequence are well known in the art. A DNA sequence encoding pp32r1,pp32r2, or an analog of either pp31R1 or pp32r2, can be synthesizedchemically or prepared from the wild-type sequence by one of severalapproaches, including primer extension, linker insertion and PCR (see,e.g., Sambrook, et al.). Mutants can be prepared by these techniqueshaving additions, deletions and substitutions in the wild-type sequence.It is preferable to test the mutants to confirm that they are thedesired sequence by sequence analysis and/or the assays described below.Mutant protein for testing may be prepared by placing the codingsequence for the polypeptide in a vector under the control of apromoter, so that the DNA sequence is transcribed into RNA andtranslated into protein in a host cell transformed by this (expression)vector. The mutant protein may be produced by growing host cellstransfected by an expression vector containing the coding sequence forthe mutant under conditions whereby the polypeptide is expressed. Theselection of the appropriate growth conditions is within the skill ofthe art.

[0067] The assembled sequence can be cloned into any suitable vector orreplicon and maintained there in a composition which is substantiallyfree of vectors that do not contain the assembled sequence. Thisprovides a reservoir of the assembled sequence, and segments or theentire sequence can be extracted from the reservoir by excising from DNAin the reservoir material with restriction enzymes or by PCRamplification. Numerous cloning vectors are known to those of skill inthe art, and the selection of an appropriate cloning vector is a matterof choice (see, e.g., Sambrook, et al., incorporated herein byreference). The construction of vectors containing desired DNA segmentslinked by appropriate DNA sequences is accomplished by techniquessimilar to those used to construct the segments. These vectors may beconstructed to contain additional DNA segments, such as bacterialorigins of replication to make shuttle vectors (for shuttling betweenprokaryotic hosts and mammalian hosts), etc.

[0068] Producing the Recombinant Peptide

[0069] Preferably, DNA from the selected clones should be subcloned intoan expression vector, and the protein expressed by cells transformedwith the vector should be tested for immunoreactivity with antibodiesagainst the recombinant protein of this invention prepared as describedbelow. Such subcloning is easily within the skill of the ordinary workerin the art in view of the present disclosure. The amino acid codingregion of the DNA sequence of this invention may be longer or shorterthan the coding region of the disclosed sequence, so long as therecombinant peptide expressed by the DNA sequence retains at least oneepitope cross-reactive with antibodies which are specificallyimmunoreactive with pp32r1, pp32r2; or other pp32 variant as desired.The preparation of selected clones which contain DNA sequencescorresponding to all or part of the sequence of pp32r1 or pp32r2 may beaccomplished by those of ordinary skill in the art using conventionalmolecular biology techniques along with the information provided in thisspecification.

[0070] It is possible to purify a pp32 variant protein such as pp32r1,which is cross-reactive with antibodies specific for pp32, from anappropriate tissue/fluid source; however, a cross-reactive pp32 variant,or analog thereof, may also be produced by recombinant methods from aDNA sequence encoding such a protein or polypeptide. Polypeptidescorresponding to the recombinant protein of this invention may beobtained by transforming cells with an expression vector containing DNAfrom a clone selected from an mammalian (preferably human) library asdescribed herein. Suitable expression vector and host cell systems arewell known to those of ordinary skill in the art, and are taught, forinstance, in Sambrook, et al., 1989. The peptide may be obtained bygrowing the transformed cells in culture under conditions wherein thecloned DNA is expressed. Of course, the peptide expressed by the clonemay be longer or shorter than pp32r1 or pp32r2, so long as the peptidesare immunologically cross-reactive. Depending on the expression vectorchosen, the peptide may be expressed as a fusion protein or a matureprotein which is secreted or retained intracellularly, or as aninclusion protein. The desired polypeptides can be recovered from theculture by well-known procedures, such as centrifugation, filtration,extraction, and the like, with or without cell rupture, depending on howthe peptide was expressed. The crude aqueous solution or suspension maybe enriched for the desired peptide by protein purification techniqueswell known to those skilled in the art. Preparation of the polypeptidesmay include biosynthesis of a protein including extraneous sequencewhich may be removed by post-culture processing.

[0071] Using the nucleotide sequences disclosed herein and thepolypeptides expressed from them, antibodies can be obtained which havehigh binding affinity for pp32r1 or pp32r2, but much lower affinity forpp32 and/or other variants of pp32. Such antibodies, whether monoclonalor purified polyclonal antibodies can be used to specifically detectpp32r1 or pp32r2. Techniques for preparing polypeptides, antibodies andnucleic acid probes for use in diagnostic assays, as well as diagnosticprocedures suitable for detection of pp32 are described in U.S. Pat.Nos. 5,726,018 and 5,734,022, incorporated herein by reference, andthese techniques may be applied to pp32r1 or pp32r2 by substitution ofthe nucleic acid sequences disclosed herein. Similar substitution may beapplied to other variants of pp32.

[0072] pp32r1 Promoter Sequence

[0073] Multiple consensus sequences for binding active steroid receptorsfound in genomic sequences upstream from the pp32r1 coding region areconsistent with hormone regulation of gene expression. The consensussequences were associated with the both induction and repression ofexpression by steroid hormones. The combination of both positively andnegatively acting elements suggests complex regulation of pp32r1expression.

[0074] Possible steroid hormone regulation of pp32r1 expression isimportant in regard to prostate cancer. While about one-half of treatedpatients initially respond to androgen ablation, subsequent hormonerefraction and continued aggressive tumor growth is common (Garnick, M.B., “Prostate Cancer,” in Scientific American Medicine, Dale, D. C. andFederman, D. D. Eds., Scientific American Inc., New York. 1995). Manydifferent steroid hormones regulate the growth of prostate cancer cells(Huggins, et al., “Studies on prostate cancer: I. The effect ofcastration, of estrogen, and of androgen injection on serum phosphatasesin metastatic carcinoma of the prostate,” Cancer Res., 1:293, 1941).These findings established a basis for androgen ablation therapy for thetreatment of metastatic prostate cancer.

[0075] The present invention provides androgen-activated promoters basedon the upstream portion of the genomic sequence in FIG. 2. The promotersequence provided by this invention is bounded at its 3′ terminus by thetranslation start codon of a coding sequence and extends upstream (5′direction) to include at least the number of bases or elements necessaryto initiate transcription at levels above background. Within thepromoter sequence will be found a transcription initiation site(conveniently defined by mapping with nuclease S1), a protein bindingdomain (consensus sequence) within about 100 bases upstream of thetranscription initiation site generally designated the TATA box (abinding site for TATA box binding proteins and RNA polymerase), andvarious other protein binding domains (consensus sequences) upstream ofthe TATA box that modulate the basic transcriptional activity of thetranscription initiation site and the TATA box. The various otherprotein binding domains preferably contain recognition sequences shownin Table 1 for binding (1) androgen receptors, estrogen receptors,glucocorticoid receptors, and progesterone receptors; (2) transcriptionfactors containing the leucine zipper motif including, but not limitedto Fos, Jun, JunB, and Myc; and, (3) certain tissue specifictranscription factors including, but not limited to GATA-1 and GATA-2.The various other protein binding domains upstream of the TATA box maycontribute to specificity (tissue specific expression), accuracy (properinitiation), and strength (transcription frequency) of the promoter. Thepromoter elements may serve overlapping functions so that the promotermay function in the absence of subsets of these elements.

[0076] Therapy

[0077] Inhibition of function of protransforming variants of pp32 by anymeans would be expected to be an avenue of therapy.

[0078] U.S. Pat. No. 5,726,018, incorporated herein by reference,describes various therapeutic avenues which may be applied by theskilled worker based on the nucleotides and protein sequences disclosedherein. In a particular embodiment, all or a portion of the sequence ofpp32r1 or pp32r2 may be supplied in the antisense orientation to blockexpression of the variants found in carcinomas particularly prostatecancer. Suitable methods for preparation of antisense expression vectorsand administration of antisense therapy may be found in U.S. Pat. No.5,756,676, incorporated herein by reference. Prescreening of the patientpopulation using the diagnostic methods described herein to identifypatients having tumors expressing the particular pp32 variant ispreferred.

[0079] Screening for compounds having therapeutic effects in prostatecancer may also be facilitated by the present invention. Studies whichmay be used to screen candidate compounds are described in U.S. Pat. No.5,756,676, incorporated herein by reference, modified by the use of celllines which express particular variants of pp32 (see, e.g., Examplesbelow). Compounds which affect steroid dependent protein expression mayalso be detected according to this invention by similar screeningstudies using an androgen-activated promoter as provided hereinoperatively coupled to a DNA sequence whose expression may be detected.(Marker sequences are well known in the art, see, e.g., Sambrook, etal., and selection of an appropriate detectable expression marker is aroutine matter for the skilled worker.) Screening by testing the effectof candidate compounds on recombinant cells containing an expressionvector having an androgen-activated promoter operatively coupled to anexpression marker, with appropriate controls, is within the skill of theart, in view of the promoter sequences provided herein. In one aspect,this invention provides a method for screening candidate compounds forpharmacological activity by (1) culturing a cell transfected with theDNA molecule containing an androgen-activated transcriptional promoterwhich is operatively linked to an open reading frame comprising at leastone exon of a protein coding sequence, and (2) determining expression ofthe open reading frame in the presence and absence of the compound. In apreferred mode the androgen activated promoter may be the portion of thesequence in FIG. 2 which is up-stream of the translation initiationsite, or alternatively the androgen activated promoter may be the 2700bp upstream from the translation initiation site.

[0080] Diagnostic Methods Based on the pp32 Gene Family

[0081] In one aspect, this invention provides methods for detecting anddistinguishing among members of the pp32 gene family. As explainedherein, the presence of one or more members of the gene family may bedetected using assays based on common structures among the membersresulting from common or similar sequences. For example, polyclonalantibodies elicited by pp32 will cross-react with pp32r1 and pp32r2,including various alleles of these pp32 variants. Similarly, the fullcoding region of the pp32 cDNA will hybridize under suitable conditionswith nucleic acid encoding any of the variants, as shown by the in situdetection of the variants in tumor sections which were subsequentlyshown to contain either pp32r1 or pp32r2 allelic forms (Example 1).Selection of conditions that promote the immune cross-reactivity orcross-hybridization necessary for such detection is within the skill ofthe art, in view of the examples provided herein. For example, by usinglarge nucleotide probes in hybridization experiments, the effects of oneor a few differences in sequence may be overcome, i.e., larger probeswill bind to more dissimilar target sequences, in contrast to shorterprobes for which each nucleotide makes a larger percentage contributionto the affinity, and a single nucleotide alteration will cause a greaterrelative reduction in hybridization efficiency. Typically probes of 50or more nucleotides are used to find homologues to a given sequence, andthe studies reported in Example 1 used the entire sequence of pp32 as aprobe to find cells expressing homologous members of the gene familyother than pp32. Likewise, polyclonal antisera elicited to an antigenhaving multiple epitopes is more likely to cross-react with a secondantigen that has a few of the same epitopes along with many differentepitopes, while a monoclonal antibody or even a purified polyclonalantiserum might not bind to the second antigen.

[0082] In addition to determining the presence of one or more members ofthe pp32 gene family, this invention also provides methods fordistinguishing among members. Determining which pp32 variant may beuseful, for instance, to determine whether a transfomation promoting orsuppressing variant is present in a tissue sample. Suitable methods fordistinguishing include both immunoassay and nucleic acid binding assays.Preferred are methods which can detect a 10-fold difference in theaffinity of the detecting ligand (e.g., antibody or oligonucleotide) forthe target analyte. Such methods are well documented for other systems,and may be adopted to distnguish between pp32 variants by routinemodification of such methods in view of the guidance provided herein.

[0083] Protein level assays may rely on monoclonal or purifiedpolyclonal antibodies of relatively greater affinity for one variantcompared to another (see, e.g., Smith, et al. (“Kinetics in interactionsbetween antibodies and haptens,” Biochemistry, 14(7):1496-1502, 1975,which shows that the major kinetic variable governing antibody-hapteninteractions is the rate of dissociation of the complex, and that thestrength of antibody-hapten association is determined principally by theactivation energy for dissociation), and Pontarotti, et al.(“Monoclonalantibodies to antitumor Vinca alkaloids: thermodynamics and kinetics,”Molecular Immunology, 22(3):277-84, 1985, which describes a set ofmonoclonal antibodies that bind various dimeric alkaloids and candistinguish among the alkaloid haptens due to different relativeaffinities of the various monoclonal antibodies for particular dimericalkaloids), each of which is incorporated herein by reference). Suitablemodifications of the conditions for immunoassays to emphasize therelative affinity of monoclonal antibodies with different affinity arealso discussed in U.S. Pat. No. 5,759,791, incorporated herein byreference.

[0084] A number of methods are available which are capable ofdistinguishing between nucleic acid sequences which differ in sequenceby as little as one nucleotide. For example, the ligase chain reactionhas been used to detect point mutations in various genes (see. e.g.,Abravaya, et al., “Detection of point mutations with a modified ligasechain reaction (Gap-LCR).” Nucleic Acids Research, 23(4):675-82, 1995,or Pfeffer, et al., “A lipase chain reaction targeting two adjacentnucleotides allows the differentiation of cowpox virus from otherOrthopoxvirus species,” Journal of Virological Methods, 49(3):353-60,1994, each of which is incorporated herein by reference). Amplificationof a sequence by PCR also may be used to distinguish sequences byselection of suitable primers, for example, short primers, preferably10-15 matching nucleotides, where at least one of the primers has on the3′ end a unique base that matches one variant but not other variants,and using annealing conditions under which the primer having the uniquebase has at least a ten-fold difference in dissociation rate between thefully matching variants and variants which do not fully match. Similardifferentiation may be achieved in other methods dependent onhybridization by using short probes (typically under 50 bp, preferably25 bp or less more preferably less than 20 bp or even 10-12 bp) byadjusting conditions in hybridization reactions to achieve at least aten-fold difference in dissociation rate for the probes between thefully matching variants and variants which do not fully match. Cleavasefragment length polymorphism may also be used, and a specific examplebelow provides guidance from which the skilled worker will be able todesign similar studies by routine selection of other cleavase enzymes inview of the sequences provided herein.

[0085] The diagnostic methods of this invention may be used forprognostic purposes and patient differentiation as described herein. Inparticular, the methods of this invention allow differentiation betweenproducts expressed from the various sequences disclosed in FIG. 7.Preferred methods are those that detect and/or differentiate, betweenpp32, pp32r1, and/or pp32r2. Situations in which differentiation betweenpp32 variants will be of benefit will be readily apparent to the skilledclinician, in view of the present disclosure. Selection among thediagnostic methods provided by this invention of a suitable technique toachieve the desired benefit is a routine matter for the skilledclinician.

EXAMPLES

[0086] In order to facilitate a more complete understanding of theinvention, a number of Examples are provided below. However, the scopeof the invention is not limited to specific embodiments disclosed inthese Examples, which are for purposes of illustration only.

Example 1

[0087] Cellular Location of pp32 Expression

[0088] pp32 mRNA can be detected by in situ hybridization with a pp32probe under stringent conditions.

[0089] In situ hybridization. Bases 1-298 of the pp32 cDNA sequence(GenBank HSU73477) were subcloned into the Bluescript vector by standardtechniques. Digoxigenin labeled anti-sense and sense RNA probes weregenerated using a commercially available kit (Boehringer Mannheim).Vector DNA linearized with BamHI and Xhol served as template forantisense and sense probe generation respectively. In vitrotranscription was performed for 2 hours at 37° in a final volume of 20μl which contained 1 μg of template DNA, 2 U/μl of either T3 or T7 RNApolymerase. 1 U/μl ribonuclease inhibitor, 1 mM each of ATP, CTP, GTP,0.65 mM UTP, 0.35 mM digoxigenin-11-UTP, 40 mM Tris-HCl pH 8.0, 10 mMNaCl, 10 mM DTT, 6 mM MgCl₂ and 2 mM spermidine. The reaction wasstopped by adding 2 μl of 0.2M EDTA, pH 8. 0 and the synthesizedtranscripts were precipitated for 30 min at −70° with 2.2 μl of 4 M LiCland 75 μl of pre-chilled ethanol. RNA was pelleted by centrifugation,washed with 80% ethanol, mildly dried and dissolved in 100 μl of DEPCtreated water. Yields of labeled probe were determined by an enzymelinked irrimunoassay using a commercially available kit (BoehringerMannheim). Non-radioactive in situ hybridization was performed withanti-sense and sense pp32 RNA probes generated by in vitro transcription(see U.S. Pat. No. 5,726,018, incorporated herein by reference). FIG. 1Ashows that normal prostatic basal cells are positive, whereas the clear,differentiated glandular cells are negative. In contrast, prostaticadenocarcinoma, shown at left, is strikingly positive. Note that thesignal is cytoplasmic since it is mRNA and not the protein that isdetected in this assay.

[0090] pp32 displays a distinctive pattern of expression in vivo (Chen,et al.; Malek. et al., “Identification and preliminary characterizationof two related proliferation-associated nuclear phosphoproteins.”Journal of Biological Chemistry, 265:13400-13409, 1990; Walensky, etal., “A novel M(r) 32,000 nuclear phosphoprotein is selectivelyexpressed in cells competent for self-renewal.” Cancer Research53:4720-4716, 1993). In normal peripheral tissues, expression isrestricted to stem-like cell populations such as crypt epithelial cellsin the gut and basal epithelium in the skin: in the adult centralnervous system, cerebral cortical neurons and Purkinje cells alsoexpress pp32. In normal prostate, basal cells express pp32, whereas pp32mRNA is not detectable by in situ hybridization in differentiatedglandular cells (FIG. 1A). In contrast, strong in situ hybridization topp32 probes is found in nearly all clinically significant humanprostatic adenocarcinomas. 87% of human prostatic adenocarcinomas ofGleason Score 5 and above express mRNA that hybridizes strongly withprobes to pp32 in contrast to only 11% of prostate cancers of GleasonScore 4 and below in a study of 55 patients.

[0091] Immunohistochemistry. Formalin-fixed, paraffin-embedded tissuewas sectioned at 4 μM, deparaffinized, hydrated, processed forheat-induced antigen retrieval at 95° in 0.01 M citrate buffer, pH 6.0,for 20 min (Cattoretti, et al., “Antigen unmasking on formalin-fixed,paraffin-embedded tissue sections,” Journal of Pathology 171:83-98,1993), then incubated overnight at room temperature with a {fraction(1/20)} dilution of anti-pp32 antibody. Following washing, the slide wassequentially developed with biotinylated swine-anti-rabbit IgG at{fraction (1/100)} (Dako), strepavidin peroxidase (Dako), anddiaminobenzidine. FIG. 1B shows a representative high-grade humanprostate cancer stained with affinity-purified rabbit polyclonalanti-pp32 antibody (Gusev, et al., “pp32 overexpression induces nuclearpleomorphism in rat prostatic carcinoma cells.” Cell Proliferation29:643-653, 1996). The left-hand panel shows a representative field at250x: the rectangle indicates the area shown in computer venerateddetail in the right-hand panel. Strongly hybridizing tumors show intenseimmunopositivity with antibodies to pp32, indicating that they expresspp32 or immunologically related proteins (FIGS. 1A and 1B).

Example 2

[0092] ESTs Corresponding to pp32

[0093] Several potential variant pp32 species have been identified inthe prostate cancer expressed sequence tag libraries of the NCI's CancerGenome Anatomy Project. Clone 588488 encodes a protein that is 96%identical to APRIL, although absent retrieval and sequencing of the fullclone, it is impossible to tell whether the entire EST clone encodes app32 related sequence; neither is it possible to assess the biologicfunction of this molecule at this time. Nevertheless, it is apparentthat the sequenced portion encodes a protein bearing great similarity topp32. This EST does not appear in the database for normal prostate. Aswith the variant pp32 species recovered from prostate cancer, generationof this molecule by mutation would require a complex mechanism.

[0094] pp32-related genes are present in other organisms. The existenceof a pp32 gene family in rodent would be consistent with the existenceof a comparably sized family in human. A murine pp32 (GenBank U73478)has 89% amino acid identity to pp32, but less identity to pp32r1 andAPRIL. (The murine cerebellar leucine rich acidic nuclear protein has asingle amino acid substitution relative to murine pp32.) We additionallyidentified a murine EST, GenBank AA066733, with closest identity toAPRIL protein at 85% identity over 148 amino acids of a predicted openreading frame. Several other murine EST's. AA212094 and W82526, areclosely related to the pp32 family but are not significantly morerelated to either pp32, pp32r1, or APRIL. A human homologue of such agene would be expected to encode a fourth member of this gene family. Weidentified EST's predicted to encode pp32-related proteins in C.elegans, schistosomes, zebrafish, and Drosophila (data not shown).However, these sequences may not represent the complete extent of thepp32 gene family in these organisms, and thus are not informative forthe likely size of the mammalian pp32 gene family.

Example 3

[0095] The Structure of a Gene Encoding a Relative of the pp32 Family

[0096] Screening a human genomic library in bacteriophages with probesgenerated from human pp32 cDNA yielded a new sequence that contained anopen reading frame encoding a protein homologous with pp32.

[0097] Screening a Human Genomic Library in Bacteriophages for pp32cDNA.

[0098] A genomic library from human placenta in the Lambda Fix II vectorwas expressed in E. coli strain XL-1 Blue MRA (Stratagene #946206).Screening for bacteriophage clones containing DNA inserts homologouswith pp32 cDNA followed routine procedures (Sambrook, et al.). Briefly,nitrocellulose filters that had overlain bacteriophage plaques werehybridized with P-32 labeled probes for pp32 cDNA. The probes wereprepared by the random primer method (Stratagene #300385) using pp32cDNA as a template (Chen, et al., Molec. Biol. Cell, 7:2045-2056,1996.).Reactive bacteriophage plaques were plugged and the bacteriophages wereeluted, reexpressed, and rescreened with pp32 cDNA probes until pure.Bacteriophage DNA was prepared by the plate lysate method (Sambrook, etal.).

[0099] Identifying Restriction Fragments within Bacteriophage DNAContaining Sequences Homologous with pp32 cDNA.

[0100] DNA from a bacteriophage clone containing pp32 cDNA sequences wasdigested with HindIII. Using routine methods, the restriction fragmentswere separated by agarose gel electrophoresis, transferred in alkalinebuffer to positively charged nylon filters, and hybridized with probesthat were selective for the 5′ and 3′ ends of the pp32 cDNA (Sambrook,et al.). The 5′ and 3′ probes were prepared as described above exceptthat the products of polymerase chain reactions (PCR) were used astemplates for the labeling reactions (Sailki, et al., Science,239:487-491, 1988.). One PCR product was a 249 base pair segment of pp32cDNA containing nucleotides 32 through 279. It was the result of areaction using a pp32cDNA template and the primers

[0101] 5′-TATGCTAGCGGGTTCGGGGTTTATTG-3′ and

[0102] 5′-GATTCTAGATGGTAAGTTTGCGATTGAGG-3′ (primer set A).

[0103] The other product was a 263 base pair segment of pp32 cDNAincluding nucleotides 677 through 938. It was the result of a reactionusing a pp32 cDNA template and the primers

[0104] 5′-GAATCTAGAAGGAGGAGGAAGGTGAAGAG-3′ and

[0105] 5′-CTATCTAGATTCAGGGGGCAGGATTAGAG-3′ (primer set B).

[0106] The PCR reactions included 35 cycles of one minute denaturationsat 95° C., one minute primer annealings at 50° C., and one minuteextensions at 72° C. (cycling program A). A 4.7 kb HindIII restrictionfragment that hybridized with the 5′ probe, but not with the 3′ probeand a 0.9 kb HindIII fragment that hybridized with the 3′ probe, but notwith the 5′ probe were subcloned into pBluescript (Gibco) by routinemethods (Sambrook, et al.). The nucleotide sequences of both strands ofpurified plasmid DNA containing the inserts were determined by automatedprocedures (DNA Analysis Facility, Johns Hopkins University School ofMedicine).

[0107] Completion of Sequencing by Direct Sequencing of PCR Products.Alignment of the sequences of the 4.7 and 0.9 kb HindIII restrictionfragments with pp32 cDNA showed about 90% homologies between the 3′ endof the 4.7 kb fragment and the 5′ region of pp32 cDNA and the 5′ end ofthe 0.9 kb fragment and the 3′ region of the pp32 cDNA. There was anunaligned 199 base pair gap of pp32 cDNA sequence between the ends ofthe restriction fragments. Primers were designed to specifically annealto relative pp32 sequences on both sides of the sequence gap. The primersequences were

[0108] 5′-GAGGTTTATTGATTGAATTCGGCT-3′ and

[0109] 5′-CCCCAGTACACTTTTCCCGTCTCA-3′ (primer set C).

[0110] Polymerase chain reactions followed cycling program A with primerset C and pure bacteriophage DNA as a template. The 943 base pairproducts were shown by ethidium bromide staining agarose gels, extractedfrom excised fragments of low melt agarose (NuSieve) electrophoresisgels, and sequenced by automated procedures as described above.

[0111] A sequence of 5,785 bases was obtained from the human placentalgenomic library bacteriophage clone containing segments homologous withpp32 cDNA (FIG. 2). This sequence was deposited in Genbank underAccession No. U71084, Locus HSU71084. The sequence has an open readingframe extending from nucleotides 4,453 to 5,154. Analysis of thenucleotide sequence upstream of the open reading frame revealedconsensus sequences for active steroid hormone receptors at over twentypositions (Table 1).

[0112] Sequence analysis of the open reading frame showed 94% sequencehomology to pp32 (FIG. 3). Alignment of the-open reading frame sequenceto pp32 cDNA revealed 33 scattered, solitary base differences andclustered differences of two and seven bases. There were two internaldeletions of three and nine bases. The open reading frame encoded apolypeptide containing 234 amino acid residues with 88% protein-levelhomology to pp32 (FIG. 4). Alignment of the translated sequence to thepp32 amino acid sequence revealed 18 scattered, solitary amino acidresidue differences, three differences in clusters of two residues, andone difference in a clusters of four residues. There were two internaldeletions of one and three residues and a terminal deletion of elevenresidues. The translated sequence contained 69 acidic residues, 26 fewerthan pp32.

Example 4

[0113] Chromosome Mapping of pp32r1

[0114] The pp32r1 gene maps to chromosome 4 as determined by PCR of theNIGMS monochromosomal panel 2 (Drwinga, et al., “NIGMS human/rodentsomatic cell hybrid mapping panels 1 and 2,” Genomics 16:311314, 1993)followed by sequencing of the PCR product. Interestingly, the fullsequence of pp32r1 including 4364 nucleotides of sequence 5′ to thestart ATG contained over 400 matches in a blastn search of thenon-redundant GenBank database. These matches were to two short regionsof about 278 and 252 base pairs (nucleotides 674-952 and 2542-2794) thatrepresent repeats in opposite orientations. The repeats aresignificantly related to elements on many chromosomes.

[0115] The human pp32 gene has been mapped to chromosome 15q22.3-q23 byfluorescence in situ hybridization (Fink, et al.). A Unigene entry forpp32 (Hs. 76689; HLA-DR associated protein 1) lists 93 EST sequencescorresponding to this gene, 12 of which contain a mapped sequence-taggedsite (STS). These STS sites are all reported to map to chromosome 15, asare many of the pp32 EST's analyzed by electronic PCR(http://www.ncbi.nlm.nih.gov). APRIL protein was also mapped tochromosome 15q25 (Mencinger, et al.; GenBank Y07969).

Example 5

[0116] Sequence Analysis of pp32r2

[0117] A pp32-related sequence (designated pp32r2) has been identifiedon chromosome 12 by methods analogous to those described in Example 2for isolation of the unique intronless pp32-related gene pp32r1, foundon chromosome 4. It was initially thought that the chromosome 12sequence, encoding a truncated protein, might represent a pseudogene;however that interpretation has been reassessed in view of the presentfindings. The sequence has been designated pp32r2, and is recorded inGenbank as locus AF008216: the sequence of pp32r2 is shown in FIG. 5. ByBESTFIT analysis (Genetics Computer Group. Inc., Wisconsin Package,version 9.1, Madison, Wis., 1997), pp32r2 is 99.5% identical to FT1.11,FT2.4 and T1, showing four nucleotide differences over the 875nucleotide overlap of the sequences: this level of variation isconsistent with a polymorphism. Similarly, BESTFIT analysis shows thatPP32R1 is 99.6 % identical to FT3.3 and 99.4% identical to FT2.2,displaying four and five nucleotide differences, respectively (see FIG.7 below).

Example 6

[0118] Sequence Comparison of Multiple Clones

[0119] Screening of a human placental genomic library in Lambda Fix IIvector (Stratagene #946206) with P-32 labeled probes for pp32 cDNAyielded a clone of approximately 23 kb. 4.7 kb and 0.9 kb HindIIIrestriction fragments of this clone hybridized with probes for pp32cDNA. The 4.7 kb clone aligned with the 5′ portion of the pp32 cDNAsequence, and the 0.9 kb fragment aligned with the 3′ end. A smalldiscontinuity of 0.2 kb was sequenced from a bridging PCR product. Nointrons were identified.

[0120] Cultured cells including the whole human embryonic line FSH173WEand the prostatic cancer cell lines PC-3 and LNCaP (American TypeCulture Collection) were grown under recommended tissue cultureconditions. Poly A+RNA was prepared by oligo dT adsorption(MicroFasTrack, Invitrogen) and used as a template for the, generationof cDNA through reactions with reverse transcriptase and random hexamers(GeneAmp RNA PCR Kit, Perkin Elmer). The cDNA sequences encoding theopen reading frame were amplified by nested PCR using primersspecifically selective for the genomic sequence over pp32 sequences. Thefinal 298 base pair products were seen by ethidium bromide stainingagarose electrophoretic gels.

[0121] Using procedures similar to those described in Example 3, exceptwithout the need for nested primers in most cases, transcripts fromDU-145 cells and from numerous patients were sequenced for comparison tothe transcripts from the above samples. The results are shown in Table2. A summary of the degree of identity between various transcripts isprovided in Table 3.

Example 7

[0122] Sequence Variation for Individual Isolates of Different CellLines and Tumor Tissue

[0123] The explanation for the apparent discordant expression of p32 incancer is that prostate tumors do not generally express pp32, but ratherexpress variant pp32 species that promote transformation, instead ofinhibiting it.

[0124] RT-PCR and CFLP. Sequences were reverse-transcribed and amplifiedusing bases 32 to 52 of HSU73477 as a forward primer and 9 19 to 938 ofthe same sequence as a reverse primer in conjunction with the TitanOne-Tube RT-PCR kit (Boehringer). Reverse transcription was carried outat 50° for 45 min followed by incubation at 94° for 2 min; thesubsequent PCR utilized 45 cycles of 92° for 45, 55° for 45 sec. and 68°for 1 min with a final extension at 68° for 10 min in a PTC 100thermocycler (MJ Research). Template RNA was isolated from cell lines orfrozen tumor samples using RNAzol B (Tel-Test) according to themanufacturer's instructions, then digested with RNAse-free DNAse 1(Boehringer). pCMV32 was used as a positive control without reversetranscription. The cleavage assay was performed according to themanufacturer's specifications (Life Technologies) with digestion at 55°for 10 min at 0.2 mM MnCl₂ and electrophoresed on a 6% denaturingpolyacrylamide sequencing gel.

[0125] At the level of RTPCR, paired normal prostate and prostaticadenocarcinoma from three patients yielded amplification products (FIG.6A) ranging from 889 to 909 bp. The reaction employed consensus primerscapable of ampliring the full-length coding sequence from pp32 and thetwo closely-related intronless genomic sequences pp32r1 and pp32r2. Thesole difference noted was a diminished amplicon yield from normal tissueas compared to neoplastic. Four human prostatic adenocarcinoma celllines, DU-145, LNCaP, PC-3, and TSUPR-1, also yielded similar products.

[0126]FIG. 6A shows RT-PCR amplified DNA from human prostate andprostate cancer cell lines. Lane a is an undigested control whose bandmigrated substantially slower than the digestion produces; samples inall other lanes were digested with cleavage as described. The lanesshow: 1 kb ladder (Lifé Technologies), A; pCMV32, B; DU-145, C; LNCaP,D; PC-3, E; TSUPr-1, F; a representative sample, FT-1, without reversetranscription, G; FN-1 H; FT-1, I; FN-2, J; FT-2, K; FN-3, L; FT3, M;negative control with template omitted. FN indicates frozen benignprostate and the number indicates the patient: FT indicates frozenprostatic adenocarcinoma and the number indicates the patient. Numberson the left-hand side of the figure indicate the size in kb of areference 1 kb DNA ladder (Life Technologies).

[0127] Qualitative differences between normal and neoplastic tissuebegan to emerge when the RT-PCR products were subcloned and analyzed bycleavage fragment length polymorphism analysis (CFLP) and sequenceanalysis. FIG. 6B shows a cleavase fragment length polymorphism analysisof cloned cDNA amplified by RT-PCR from human prostatic adenocarcinoma,adjacent normal prostate, and human prostatic adenocarcinoma cell linesusing primers derived from the normal pp32 cDNA sequence. The lanes showindividual RT-PCR-derived clones from the DU-145, LNCaP, PC-3 and TSUPr1cell lines, from frozen prostate cancer (FT), and from frozen normalprostate (FN): a, undigested normal pp32 cDNA, be normal pp32cDNA: c,DU-145-1; d, DU-145-3; e, DU-145-5; f, LNCaP-3; g, PC3-3; h, PC3-8; i,TSUPr1, -I; j, TSUPr1-3; k, TSUPr1-6; 1, FT1.11; m, FT1.7; n, FT2.2; o,FT2.4; p, FT3.18; q, FT3.3; r, FN3.17; s, FN2.1. LNCaP expresses normalpp32. The band shifts correspond to sequence differences. All clones ofRT-PCR product from normal prostate tissue displayed a normal CFLPpattern that corresponded precisely to that obtained from cloned pp32cDNA template (GenBank HSU73477, FIG. 6B). Prostatic adenocarcinomasyielded four distinct CFLP patterns upon similar analysis, of whichthree were unique and one mimicked the normal pp32 pattern. Examinationof DU-145, PC-3, and TSUPR-1 cell lines yielded substantially similarresults whereas LnCaP yielded only a normal pp32 CFLP pattern. Furtheranalysis at the sequence level confirmed that normal prostate and LnCaPcontained solely normal pp32 transcripts.

[0128] Transcripts obtained from prostatic adenocarcinomas and from mostcell lines represented closely-related variant species of pp32,summarized in Table 1. These transcripts varied from 92.4% to 95.9%nucleotide identity to normal pp32 cDNA (Genetics Computer Group, Inc.,Wisconsin Package, version 9.1, Madison, Wis., 1997). Of the sixteenvariant transcripts obtained, fifteen had open reading frames encodingproteins ranging from 89.3% to 99.6% identity to normal pp32. The tablesummarizes data obtained for variant pp32 transcripts obtained fromhuman prostatic adenocarcinoma and prostate cancer cell lines. Sequencesfalling into closely related groups are indicated by the group letters(A,B,C); U indicates unassigned sequences not clearly falling into agroup. The origin of each sequence is: FT, frozen tumor followed bypatient number, decimal point, and clone number; D, DU-145 followed byclone number (as are all cell line sequences); P, PC3; and T, TSUPr1.Nucleotide identity, gaps in the nucleotide sequence alignment, andprotein identity were determined from BESTFIT alignments with the normalpp32 cDNA and protein sequences. The effect on transformation isdescribed as: stimulates, more foci obtained when transfected withras+myc than with ras-myc+vector control: inactive, equivalent fociobtained as with ras+myc+vector control; and suppresses, fewer fociobtained as with ras+myc+vector control.

[0129] The predicted protein sequences fell into three discrete groups:[1] truncated sequences spanning the N-terminal 131 amino acids of pp32,of which one such sequence substantially equivalent to pp32r2 wasobtained identically from two of three patients and from the TSUPR-1cell line; [2] sequences more closely homologous to a distinctpp32-related gene, pp32r1 than to pp32, and [3] heterogeneouspp32-related sequences. Tumors from two of the three patients analyzedcontained no detectable normal pp32 transcripts. Two of twelve clonedtranscripts from the third patient tumor were normal by CFLP pattern,with sequence confirmation of normality on one clone. Two clones fromcell lines were normal by CFLP screening, but were later shown torepresent variant-sequences.

[0130]FIGS. 7A and 7B show a multiple pairwise alignment of nucleotideand predicted protein sequences for all transcripts (Smith, et al.,“Identification of common molecular subsequences,” J. Mol. Biol.,147:195-197 1981). The figures were compiled with the GCG Pileup andPretty programs (Smith, et al.). Differences from the consensussequences are shown as indicated, agreement with the consensus sequenceis shown as a blank. Normal human pp32 is designated hpp32. Sequencesfrom the TSUPr1, PC3, and DU-145 cell lines are as indicated. Thedesignation FT indicates sequence derived from a frozen human prostaticadenocarcinoma. Only the normal pp32 sequence. hpp32, was obtained fromnormal prostate adjacent to tumor tissue. FIG. 8A shows alignment of theamplicon nucleotide sequences with pp32 and the predicted amplicon frompp32r1: FIG. 8B shows alignment of the predicted protein sequences. Onesequence (FT 1.11), independently obtained three times from two separatepatients and the TSUPR-1 cell line, is shown only once in the diagram.The pileup and pairwise alignments illustrate several important points:[1] there is a high degree of sequence conservation at both thenucleotide and predicted amino acid levels; [2] the sequence differencesare distributed throughout the length of the sequence without obvioushotspots; [3] there is no obvious clustering or segmentation of sequencedifferences: and [4] the variant sequences fall into the previouslydescribed groups. These points are detailed in FIGS. 8A and 8B.

Example 8

[0131] Diagnostic Method to Distinguish Among Family Members

[0132] The three members of the pp32 family which are expressed in humanprostate cancer are pp32, pp32r1 and pp32r2. Whereas pp32 suppresses invitro transformation and in vivo tumorigenesis in model systems, pp32r1and pp32r2 are pro-transforming and are tumorigenic in the same systems.It is possible to determine which of the three members is expressed in atissue sample by using a protocol similar to that described in Example7.

[0133] Analysis from freshly frozen human tissue and cell lines. TotalRNA is extracted from freshly frozen human tissues or human cancer celllines and subjected to reverse transcription and polymerase chainreaction amplification with single set of primers capable of amplifyingthe entire coding region of the cDNA of all the three genes. A suitableset of primers is:

[0134] Upper: 5′GGGTTCGGGGTTTATTG3′—This corresponds to bp32 to bp48 ofthe pp32 cDNA sequence (Genbank U73477)

[0135] Lower: 5′CTCTAATCCTGCCCCCTGAA3′—This corresponds to bp919 tobp938 of the pp32 cDNA sequence (Genbank U73477)

[0136] The observed amplicon sizes with this primer set are pp32-907 bp,pp32r1-889 bp and pp32r2-900 bp. The three cDNAs are distinguished fromeach other by restriction enzyme digestion with the followingenzymes—EcoR I, Hind III and Xho I. The resultant digest is run on a2.5% agarose gel to positively identify the three different cDNAs. Thetable below lists the sizes of the bands observed The bolded numbersindicate the band sizes useful for identification of the three cDNAs.TABLE 4A Expected band sizes upon restriction digestion of the RT-PCRproduct from fresh tissue and cell lines EcoR I/Hind III EcoR I/Xho IUndigested EcoR I Double digest Double digest hpp32 907 21,177,70921,177,69,640 21,177,709 pp32r1 889 21,177,691 21,19,66,198,42721,177,691 pp32r2 900 21,879 21,244,635 21,385,494

[0137] Analysis from formalin fixed and paraffin embedded tissue. Asimilar approach is followed for identification of pp32, pp32r1 andpp32r2 transcripts from formalin fixed and paraffin embedded tissues.Total RNA is extracted and subjected to reverse transcription and PCRamplification with a single set of primers capable of amplifying astretch of 200 bp from all the three cDNAs. A suitable set of primersis:

[0138] Upper primer—from bp394 to bp414 of the pp32 cDNA sequence(Genbank U73477)

[0139] Lower primer—from bp609 to bp629 of the pp32 cDNA sequence(Genbank U73477)

[0140] The three cDNAs are distinguished from each other by restrictionenzyme digestion with the following enzymes—Hind III, Xho I and BseR 1.The resultant digest is run on a 3% agarose gel to positively identifythe three different cDNAs. The table below lists the sizes of the bandsobserved. The bolded numbers indicate the band sizes useful foridentification of the three cDNAs. TABLE 5A Expected band sizes uponrestriction digestion of the RT-PCR product from formalin fixed andparaffin embedded tissues Undigested Hind III Xho I BseR I hpp32 200 200200 80,120 pp32r1 200 100,100 200 200 pp32r2 200 200 44,156 80,120

Example 9

[0141] pp32r1 Augments Oncogene-Mediated Transformation of Rat EmbryoFibroblasts.

[0142] pp32r1 was subcloned into a eukaryotic expression vector underthe CMV promoter and analyzed for its effect on ras+myc-mediatedformation of transformed foci in rat embryo fibroblasts. Genomicsequences including the entire coding region for pp32r1 were amplifiedby PCR and subcloned into the eukaryotic TA cloning and expressionvector pCR3.1 vector (Invitrogen) which contains a CMV promoter. Theassay was performed as described (Chen et al. Mol Biol Cell, 7:2045-56,1996) with each T75 flask receiving 5 micrograms of pEJ-ras, and/or 10micrograms of pMLV-c-myc, pCMV32, pp32r1 in PCR3.1, or PCR 3.1 alone.After 14 days, transformed colonies were enumerated. FIG. 8 shows theresults. The data represent the average of seven replicates from twoseparate experiments in duplicate and one in triplicate. The error barsindicate standard error of the mean. In contrast to pp32, whichconsistently suppresses focus formation induced by ras+myc and otheroncogene pairs, pp32r1 caused a statistically significant stimulation offocus formation with p=0.004 by an unpaired t-test.

Example 10

[0143] Effect of Transcripts from Various Cell Lines on Rat FibroblastTransformation Assays

[0144] Expression constructs prepared as described above from PC-3 andDU-145 cells were tested in the rat embryo fibroblast transformationassay described by Chen, et al., Mol Biol Cell., 7:2045-56, 1996,incorporated herein by reference. The results are shown in FIG. 9.Transcripts from the two cell lines stimulated ras+myc induction oftransformed rat embryo fibroblast foci, in contrast to normal pp32,which suppressed transformation. The figure shows the mean±the standarddeviation, except for DU-145, which represents a single determination.

Example 11

[0145] Transformation Activity of Various Isolates from Patient Tumors

[0146] The variant transcripts isolated from prostate cancer patientsdiffer significantly from pp32 in sequence. The isolated transcriptswere found to stimulate transformation. Transformation assay. Rat embryofibroblasts were transfected with the indicated constructs as described(Chen, et al.) and transformed foci enumerated. For each experiment,approximately 1×10⁶ cells were plated per T75 flask and incubated for 2to 3 d prior to transfection to achieve approximately 40% confluency.For each flask of primary rat embryo fibroblasts, the plasmids indicatedin each experiment were added in the following amounts: pEJ-ras, 5 μg;and pMLV-c-myc, pCMV32, pCMVneo, or variant pp32 constructs in pCR3.1(Invitrogen), 10 μg. Plasmids were prepared in two volumes Lipofectin (2μl lipofectin per μg DNA) then gently mixed by inversion in 1.5 mlOPTIMEM in sterile 15 ml polystyrene tubes and allowed to incubate atroom temperature for >15 min. For experiments with more than one flask,mixtures of all reagents were increased in proportion to the numbers offlasks required for each transfection. Cells were washed once withOPTIMEM (Gibco-BRL), and then fed with 6 ml of OPTIMEM and 1.5 ml of theDNA/Lipofectin mix. After overnight incubation, the cells were grown instandard media and refed with fresh media twice weekly. Foci werecounted fourteen days post-transfection. FIG. 10 summarizes fourseparate experiments. Each data point represents the results from anindividual flask expressed as the percent foci obtained in thecontemporaneous control of ras+myc+vector.

[0147]FIG. 10 shows that expressed variant transcripts from prostatecancer cell lines and from human prostatic adenocarcinoma generallyproduce increased numbers of transformed foci when co-transfected withras and myc as compared to the number of foci obtained when ras and mycare transfected with blank vector. Variant pp32 transcripts from DU-145(D3), and from three prostate cancers (FT 1.7, FT 2.2 and FT3.18) yieldincreased numbers of transformed foci over those produced by ras and mycalone with blank vector. This stands in marked contrast to normal pp32,which consistently suppresses transformation. These activities are alsosummarized in Table 1.

Example 12

[0148] Effect of pp32 Variants on Tumorigenesis in Vivo

[0149] Experiments testing the effect of transfection of NIH3T3 cells ontumorigenesis in vivo are consistent with in vitro results in rat embryofibroblasts. NIH3T3 cells were stably transfected by lipofection withthe pp32 species indicated in Table 6A carried in the pCR3.1-UniCMV-driven mammalian expression vector (Invitrogen). The G418-resistantclones employed in these experiments were all shown by genomic PCR tocarry the indicated pp32 species. For analysis of tumorigenesis, 5×10⁶cells in 100 microliters of unsupplemented Dulbecco's modified Eagle'smedium without phenol red were injected into the flanks of femaleathymic nude mice on an outbred background of greater than six weeks inage (Harlan). For logistical reasons, inoculations of the various groupswere staggered over a seven day period. Each group of mice waseuthanized precisely seven weeks after inoculation. Where a mouse had atumor, the tumor was dissected, measured, and weighed, and Table 6Areports the average weight of tumors in mice injected with cellscarrying various vectors. One tumor from each group was examinedhistologically. All tumors were fibrosarcomas without noteworthyinflammation present. Data obtained with NIH3T3 cells indicate thatNIH3T3 cells stably transfected with the variant pp32 species P3, P8,FT1.7, FT2.2, and FT2.4 form tumors when inoculated into nude mice. Incontrast, NIH3T3 cells stably transfected to express human pp32 fail toform tumors in vivo even when further transfected with ras. Lines ofNIH3T3 cells were also established that were stably transfected withexpression constructs encoding pp32 or pp32-antisense. Basal expressionof pp32 is essential for maintenance of contact inhibition andserum-dependent cell growth: antisense ablation of endogenous pp32synthesis permitted cells to grow normally following serum withdrawal.Constitutive over-expression of pp32 potently suppressed ras-mediatedtransformation of NIH3T3 cells in vitro and tumorigenesis in vivo. Incontrast, antisense ablation of endogenous pp32 dramatically increasedthe number and size of ras-transformed foci; in vivo, tumors obtainedfrom ras-transformed antisense pp32 cells were approximately 50-foldgreater in mass than tumors obtained from ras-transformed control cells.

[0150] For purposes of clarity of understanding, the foregoing inventionhas been described in some detail by way of illustration and example inconjunction with specific embodiments, although other aspects,advantages and modifications will be apparent to those skilled in theart to which the invention pertains. The foregoing description andexamples are intended to illustrate, but not limit the scope of theinvention. Modifications of the above-described modes for carrying outthe invention that are apparent to persons of skill in medicine,immunology, hybridoma technology, pharmacology, pathology, and/orrelated fields are intended to be within the scope of the invention,which is limited only by the appended claims.

[0151] All publications and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains. All publications and patentapplications are herein incorporate reference to the same extent as ifeach individual publication or patent application was specifically andindividually indicated to be incorporated by reference. TABLE 1Consensus Position Strand Sequence Factor 4 C TTTCCT PEA3 21 N CAAGGTCAELP 23 N AGGTCA PPAR 32 C CCCTAA TBF1 41 N CTTGGC NF-1 (-like proteins)81 N TAAACAC Pit-1 82 N AAACACA HiNF-A 113 C CTTCCC c-Ets-2 118 N CTATCAGATA-1 122 N CAGTTG c-Myc 212 C AATAAATA TFIID 213 N ATAAATA ETF 247 NTATCTA NIT2 261 C AAGGAA c-Ets-2 262 N AGGAAA PEA3 283 C TTTTTCTTTTTC Hb320 C TTATAT GAL4 333 N TAAAAAA TBP 349 N TTATACATT TBP 363 C AAGGAAc-Ets-2 394 C TTTCTATA TBP 398 N TATAAA TBP 398 N TATAAA TFIID 411 CCTGAATT Pit-1 420 N TGTCCC GR 423 C CCCTAA TBF1 434 N TTCCTT c-Ets-2 447C CTTCCC c-Ets-2 514 N TTATCTCT GATA-1 514 C TTATCT GATA-2 515 N TATCTCNIT2 537 N TATGCA EFII 553 N AAGTCA GCN4 608 N TGACTA GCN4 628 NCCTCCCAAC LyF-1 640 N TGTCCT GR 648 N TTAAAATTCA 1-Oct 648 N TTAAAATTCA4-Oct 649 N TAAAAT F2F 649 N TAAAAT Pit-1 661 N TAAAAAA TBP 673 N CTTGGCNF-1 (-like proteins) 725 N AGGCGG Sp1 729 N GGGCGG ETF 729 N GGGCGG Sp1729 C GGGCGG Sp1 741 N AGGTCA PPAR 793 N TATAAATA B factor 793 N TATAAATBP 793 N TATAAATA TFIID 793 N TATAAAT TMF 794 N ATAAATA ETF 809 NTTATCT GATA-1 809 C TTATCT GATA-2 815 N GGGTGTGG TEF-2 826 C CACATGmuEBP-C2 826 C CACATG TFE3-S 826 N CACATG USF 978 N ATGTAAAACA 1-Oct 978N ATGTAAAACA 2-Oct 978 N ATGTAAAACA NF-IL-2A 1000 N ATGTCAGA CSBP-1 1006N GATTTC H4TF-1 1034 C TTTTCAT Pit-1 1047 N AAGATAAAACC RVF 1048 CAGATAA GATA-1 1048 N AGATAA GATA-2 1049 N GATAAA TFIID 1083 C GCCAAGNF-1 (-like proteins) 1124 N CGCCAT UCRF-L 1163 C GACCTG TGT3 1307 NCAGTCA GCN4 1347 C TGCATA EFII 1373 C AGAACA AR 1373 N AGAACAT GR 1373 NAGAACA GR 1373 C AGAACA GR 1373 N AGAACA PR 1373 C AGAACA PR 1373 NAGAACA PR A 1373 C AGAACA PR A 1393 C TCACTT IRF-1 1393 C TCACTT IRF-21395 C ACTTCCT E1A-F 1423 N TTATCT GATA-1 1423 C TTATCT GATA-2 1424 NTATCTA NIT2 1452 N TTACTC GCN4 1471 N TGGGTCA c-Fos 1471 N TGGGTCA c-Jun1471 N TGGGTCA ER 1496 N TCTCTTA c-Myc 1511 N TATAAA TBP 1511 N TATAAATFIID 1549 C TTTGAA TFIID 1568 C AATGTATAA TBP 1581 C TTTGAA TFIID 1590C AGATAA GATA-1 1590 N AGATAA GATA-2 1591 C GATAATTG Dfd 1657 C AGGACAGR 1670 C ATTTTA F2F 1670 C ATTTTA Pit-1 1671 C TTTTATA B factor 1671 CTTTTATA Dr1 1671 C TTTTATA En 1671 C TTTTATA TBP 1671 C TTTTATA TBP-11671 C TTTTATA TFIIA 1671 C TTTTATA TFIIB 1671 C TTTTATA TFIID 1671 CTTTTATA TFIIE 1671 C TTTTATA TFIIF 1671 C TTTTATA TRF 1672 C TTTATA TBP1694 C AATAAATA TFIID 1695 N ATAAATA ETF 1733 N AGGAAA PEA3 1749 CTTATAT GAL4 1783 N TAACTCA AP-1 1829 C TAGATA NIT2 1857 N CGCCAT UCRF-L1875 N TTCTGGGAA IL-6 RE-BP 1895 N TGACTA GCN4 1899 N TATTTAA TBP 1942 NATATAA GAL4 1985 C TTTATA TBP 1985 C TTTATA TFIID 2010 C AATAAATA TFIID2011 N ATAAATA ETF 2058 C TGCATA EFII 2095 N CAGTCA GCN4 2146 C AAGGAAc-Ets-2 2147 N AGGAAA PEA3 2190 N AGGAAA PEA3 2220 C GGCACA GR 2252 NCCAATAG gammaCAAT 2286 N TGTGCC GR 2292 N ATGGGA PTF1-beta 2314 N TATGCAEFII 2328 C GGCACA GR 2350 C ATGATAAG GATA-1 2351 N TGATAAG GATA-1 2363N GGGAAG c-Ets-2 2367 N AGCCACT CP2 2369 C CCACTGGGGA AP-2 2404 N TAAAATF2F 2404 N TAAAAT F2F 2404 N TAAAAT Pit-1 2409 N TTGTCATA 77 + 82Kprotein 2409 N TTGTCATA VETF 2415 N TATCTA NIT2 2451 C TTTATC TFIID 2452N TTATCT GATA-1 2452 C TTATCT GATA-2 2486 N CTCTCTCTCTCTC GAGA factor2644 N AGGCGG Sp1 2658 N ACAGCTG GT-IIBalpha 2658 N ACAGCTG GT-IIBbeta2709 C GGCCAGGC AP-2 2723 N TGAACT GR 2731 C TGACCT PPAR 2731 C TGACCTCAURTF 2753 N CTTGGC NF-1 (-like proteins) 2818 C TGATGTCA AP-1 2818 CTGATGTCA c-Fos 2818 C TGATGTCA c-Jun 2818 C TGATGTCA CREB 2845 N GGGAAGc-Ets-2 2858 N AGATAG GATA-1 2858 C AGATAG GATA-1 2864 C AGTTCA GR 2899N ATATAA GAL4 2900 N TATAAAA B factor 2900 N TATAAAA Dr1 2900 N TATAAAAEn 2900 N TATAAAA TBP 2900 N TATAAA TBP 2900 N TATAAAA TBP-1 2900 NTATAAAA TFIIA 2900 N TATAAAA TFIIB 2900 N TATAAAA TFIID 2900 N TATAAAATFIIE 2900 N TATAAAA TFIIF 2900 N TATAAAA TRF 2921 C TTTGAA TFIID 2924 CGAAATC H4TF-1 2930 C CATTAG Is1-1 2948 C TGTACA GR 2948 C TGTACA PR 2948C TGTACA PR A 2964 C ATTTGAGAA VITF 3030 N AGTGTTCT GR 3032 N TGTTCT AR3032 N TGTTCT GR 3032 C TGTTCT GR 3032 N TGTTCT PR 3032 C TGTTCT PR 3032N TGTTCT PR A 3032 C TGTTCT PR A 3104 C GGATTATT T11 3106 C ATTATTAAAFP1 3111 N TAAAAT F2F 3111 N TAAAAT Pit-1 3125 C ATTTTA F2F 3125 CATTTTA Pit-1 3142 N TGTGAT GR 3169 N GTTTTATT HOXD10 3169 N GTTTTATTHOXD8 3169 N GTTTTATT HOXD9 3175 C TTTGAA TFIID 3185 N TTGCTCA Zta 3206N GATTTC H4TF-1 3212 N AGGAAA PEA3 3238 C ATTTTA F2F 3238 C ATTTTA Pit-13256 C TTTGAA TFIID 3266 N TTGCTCA Zta 3320 C ATTTTA F2F 3320 C ATTTTAPit-1 3358 N ATGGGA PTF1-beta 3360 C GGGACA GR 3440 C CACTCA GCN4 3460 CTTTCCT PEA3 3483 N GACACA GR 3491 C TTTCCT PEA3 3495 N CTAATG Is1-l 3523C AGAACA AR 3523 N AGAACA GR 3523 C AGAACACT GR 3523 C AGAACA GR 3523 NAGAACA PR 3523 C AGAACA PR 3523 N AGAACA PR A 3523 C AGAACA PR A 3538 CTTTATC TFIID 3539 N TTATCT GATA-1 3539 C TTATCT GATA-2 3551 N TGAGTGGCN4 3569 C TCCCAT PTF1-beta 3594 N TTAGGG TBF1 3653 C CCTGCTGAA LyF-13668 N CTCATGA 1-Oct 3668 N CTCATGA 2-Oct 3668 N CTCATGA Oct-2B 3668 NCTCATGA Oct-2B 3668 N CTCATGA Oct-2C 3679 C TGTGTAA Zta 3685 C AGAACT GR3712 C TTTCCT PEA3 3713 N TTCCTT c-Ets-2 3717 N TTGCTCA Zta 3727 CAAAACATAAAT ssARS-T 3749 N TAAAAAA TBP 3784 C CACTCA GCN4 3791 C ATTTTAF2F 3791 C ATTTTA Pit-1 3815 N TATCTA NIT2 3829 C TAGATA NIT2 3859 CAGAACA AR 3859 N AGAACAG GR 3859 N AGAACA GR 3859 C AGAACA GR 3859 NAGAACA PR 3859 C AGAACA PR 3859 N AGAACA PR A 3859 C AGAACA PR A 3860 NGAACAG LVa 3877 C ATCACA GR 3886 N TGAGTCA AP-1 3886 C TGAGTCA AP-1 3886C TGAGTCA c-Fos 3886 C TGAGTCA c-Jun 3886 C TGAGTCA FraI 3886 C TGAGTCANF-E2 3887 C GAGTCA GCN4 3931 N AGATAG GATA-1 3931 C AGATAG GATA-1 3960N TTGGCA NF-1/L 3965 C ATTTTA F2F 3965 C ATTTTA Pit-1 4026 N TATTTAA TBP4037 N TGTGAT GR 4040 N GATGCAT Pit-1 4Q42 C TGCATA EFII 4079 N TTCAAAGSRY 4079 N TTCAAAG TCF-1A 4079 N TTCAAA TFIID 4097 N CAGGTC TGT3 4140 NTGATTCA AP-1 4140 C TGATTCA AP-1 4140 N TGATTC GCN4 4164 N GGGAGTG p3004205 C AGATAA GATA-1 4205 N AGATAA GATA-2 4219 C TTAGTCAC AP-1 4219 CTTAGTCA AP-1 4219 C TTAGTCAC c-Fos 4219 C TTAGTCAC c-Jun 4219 C TTAGTCAc-Jun 4219 C TTAGTCA Jun-D 4220 C TAGTCA GCN4 4271 N TGTTCT AR 4271 NTGTTCT GR 4271 C TGTTCT GR 4271 N TGTTCT PR 4271 C TGTTCT PR 4271 NTGTTCT PR A 4271 C TGTTCT PR A 4280 C TGACCCA c-Fos 4280 C TGACCCA c-Jun4280 C TGACCCA ER 4292 C CTTATCAG GATA-1 4292 C CTTATCA GATA-1 4361 NTTCAAAG SRY 4361 N TTCAAAG TCF-1A 4361 N TTCAAA TFIID

[0152] TABLE 2 COMPARISON OF ALL PROTEIN SEQUENCES 1 15 16 30 31 45 4660 61 75 TSU6 MEMGRRIHLELRNGT PSDVKELVLDNSRSN EGKLEGLTDEFEELEFLSTINVGLTSIANL PKLNKLKKLELSSNR D3 MEMGRRIHLELRNRT PSDVKELVLDNSRSNEGKLEGLTDEFEELE FLSTINVGLTSIANL PKLNKLKKLELSDNR PG MEMGKWIHLELRNRTPSDVKELFLDNSQSN EGKLEGLADEFEELE LLNTINIGLSSIANL AKLNKLKKLELSSNR FT1.11MEMGKWIHLELRNRT PSDVKELFLDNSQSN EGKLEGLTDEFEELE LLNTINIGLTSIANLPKLNKLKKLELSSNR TSU1 MEMGKWIHLELRNRT PSDVKELFLDNSQSN EGKLEGLTDEFEELELLNTINIGLTSIANL PKLNKLKKLELSSNR FT3.18 MEMGKWIHLELRNRT PSDVKELFLDNSQSNEGKLEGLTDEFEELE LLNTINIGLTSIANL PKLNKLKKLELSSNR FT2.4 MEMGKWIHLELRNRTPSDVKELFLDNSQSN EGKLEGLTDEFEELE LLNTINIGLTSIANL PKLNKLKKLELSSNR FT2.2MEMGRRIHSELRNRA PSDVKELVLDNSRSN EGKLEALTDEFEELE FLSKINGGLTSISDLPKL-KLRKLEL---K KG MEMGRRIHSELRNRA PSDVKELALDNSRSN EGKLEALTDEFEELEFLSKINGGLTSISDL PKL-KLRKLEL---R FT1.7 MEMGRRIHLELRNRT PSDVKELVLDNSRSNEGKLEGLTDEFEELE FLSTINVGLTSIANL PKL-KLRKLEL---R P3 MEMGKWIHLELRNRTPSDVKELFLDNSQSN EGKLEGLTDEFEELE LLNTINIGLTSIANL PKLNKLKKLELSSNR L3MEMGRRIHLELRNRT PSDVKELVLDNSRSN EGKLEGLTDEFEELE FLSTINVGLTSIANLPKLNKLKKLELSDNR pp32 MEMGRRIHLELRNRT PSDVKELVLDNSRSN EGKLEGLTDEFEELEFLSTINVGLTSIANL PKLNKLKKLELSDNR P8 MEMGRRIHLELRNRT PSDVKELVLDNSRSNEGKLEGLTDEFEELE FLSTINVGLTSIANL PKLNKLKKLELSSNR 76 90 91 105 106 120 121135 136 150 TSU6 ASVGLEVLAEKCPNL 90 IHLNLSGNKIKDLST IEPLKKLENLESLDLFTCEVTNLNNY---- --------------- D3 VSGGLEVLAEKCPNL 90 IHLNLSGNKIKDLSTIEPLKKLENLESLDL FTCEVTNLNNY---- --------------- PG ASVGLEVLAEKCPNL 90IHLNLSGNKIKDLST IEPLKKLENLESLDL FTCEVTNLNNY---- --------------- FT1.11ASVGLEVLAEKCPNL 90 IHLNLSGNKIKDLST IEPLKKLENLESLDL FTCEVTNLNNY------------------- TSU1 ASVGLEVLAEKCPNL 90 IHLNLSGNKIKDLST IEPLKKLENLESLDLFTCEVTNLNNY---- --------------- FT3.18 ASVGLEVLAEKCPNL 90IHLNLSGNKIKDLST IEPLKKLENLESLDL FTCEVTNLNNY---- --------------- FT2.4ASVGLEVLAEKCPNL 90 IHLNLSGNKIKDLST IEPLKKLENLESLDL FTCEVTNLNNY------------------- FT2.2 VSGGLEVLAEKCPNL 86 THLYLSGNKIKDLST IEPLKQLENLKSLDLFNCEVTNLNDYGENV FKLLLQLTYLDSCYW KG VSGGLEVLAEKCPNL 86 THLYLSGNKIKDLSTIEPLKQLENLKSLDL FNCEVTNLNDYGENV FKLLLQLTYLDSCYW FT1.7 VSGGLEVLAEKCPNL 86THLYLSGNKIKDLST IEPLKQLENLKSLDL FNCEVTNLNDYGENV FKLLLQLTYLDSCYW P3ASVGLEVLAEKCPNL 90 IHLNLSGNKIKDLST IEPLKKLENLESLDL FTCEVTNLNNYRENVFKLLPQLTYLDGYDR L3 VSGGLEVLAEKCPNL 90 THLNLSGNKIKDLST IEPLKKLENLESLDLFNCEVTNLNDYRENV FKLLPQLTYLDGYDR pp32 VSGGLEVLAEKCPNL 90 THLNLSGNKIKDLSTIEPLKKLENLESLDL FNCEVTNLNDYRENV FKLLPQLTYLDGYDR P8 ASVGLEVLAEKCPNL 90IHLNLSGNKIKDLST IEPLKKLENLESLDL SNCEVTNLNDYRENV FKLLPQLTYLDGYDR 151 165166 180 181 195 196 210 TSU6 --------------- --------------- 131--------------- --------------- D3 --------------- --------------- 131--------------- --------------- PG --------------- --------------- 131--------------- --------------- FT1.11 --------------- ---------------131 --------------- --------------- TSU1 --------------- ---------------131 --------------- --------------- FT3.18 ------------------------------ 131 --------------- --------------- FT2.4--------------- --------------- 131 --------------- ---------------FT2.2 DHKEAPYSDIEDHVE GLDDEEEGEHEEEYD 176 EDAQVVEDEEGEEEEEEGEEEDVSGGDEED KG DHKEAPYSDIEDHVE GLDDEEEGEHEEEYD 176 EDAQVVEDEEGEEEEEEGEEEDVSGGDEED FT1.7 DHKEAPYSDIEDHVE GLDDEEEGEHEEEYD 176EDAQVVEDEEGEEGE EEGEEEDVSGGDEED P3 DDKEAPDSDAEGYVE GLDDEEEDEDEEEYD 180EDAQVVEDEEDEDEE EEGEEEDVSGEEEED L3 DDKEAPDSDAEGYVE GLDDEEEDEDEEEYD 180EDAQVVEDEEDEDEE EEGEEEDVSGEEEED pp32 DDKEAPDSDAEGYVE GLDDEEEDEDEEEYD 180EDAQVVEDEEDEDEE EEGEEEDVSGEEEED P8 DDKEAPDSDAEGYVE GLDDEEEDEDEEEYD 180EDAQVVEDEEDEDEE EEGEEEDVSGEEEED 211 225 226 240 241 TSU6 ------------------------------ --------- 131 D3 --------------- ------------------------ 131 PG --------------- --------------- --------- 131 FT1.11--------------- --------------- --------- 131 TSU1 ------------------------------ --------- 131 FT3.18 --------------- ------------------------ 131 FT2.4 --------------- --------------- --------- 131 FT2.2EEGYNDGEVDGEEDE EELGEEERGQKRK-- --------- 234 KG EEGYNDGEVDGEEDEEELGEEERGQKRK-- --------- 234 FT1.7 EEGYNDGEVDDEEDE EELGEEERGQKRKREPEDEGEDDD 245 P3 EEGYNDGEVDDEEDE EELGEEERGQKRKRE PEDEGEDDD 249 L3EEGYNDGEVDDEEDE EELGEEERGQKRKRE PEDEGEDDD 249 pp32 EEGYNDGEVDDEEDEEELGEEERGQKRKRE PEDEGEDDD 249 P8 EEGYNDGEVDDEEDE EELGEEERGQKRKREPEDEGEDDD 249

[0153] TABLE 3 Comparison to pp32 Sequences % Identity % SimilarityCLONE cDNA Protein Protein D3, DU-145 cells 95 90 95 P3, PC-3 86 94 96P8, PC-3 98 97 97 FT1.11 97 86 92 FT1.7 95 95 95 FT2.2 94 85 88 FT2.4 9986 92 FT3.18 99 90 94

[0154] TABLE 1A Effect on Oncogene- Sequence Nucleotide Protein IdentityMediated Sequence Group Identity with pp32 Gaps with pp32 TransformationComment FT1.3 A 99.8 100 Not Tested Identical to pp32 D1 A 99.9 100 Nottested identical to pp32 with 2 silent nt changes L3 A 99.9 100 NotTested D3 U 95.8 0 96.9 Generally Encodes truncated variant Stimulatorypp32 D5 U 99.6 0 99.6 Not Tested FT1.2 U 92.9 1 Not tested No ORF P3 U96.5 1 94.4 Slightly Stimulatory P8 U 98.7 0 98.0 Variable FT1.11 B 92.42 89.3 Not Tested All sequences identical, appears to be product ofpp32r2 FT2.4 B 92.4 2 89.3 Variable T1 B 92.4 2 89.3 T6 U 94.2 1 93.9Not Tested Encodes truncated variant pp32 FT3.18 U 94.7 2 89.3Stimulatory Encodes truncated variant pp32 FT2.2 C 94.4 3 87.6Stimulatory Sequences differ by 1 nt appears to be product of pp32r1FT3.3 C 94.4 3 87.6 not tested FT1.7 U 95.9 2 91.4 Stimulatory

[0155] TABLE 2A Genbank Protein Accession Length Human pp32 Human pp32r1Human pp32r2 Human April Murine pp32 Human pp32 HSU73477 249 100% 88%Identity 84% Identity 0 71% Identity 89% Identity 2 gaps; Z = 77 gaps; Z= 73 3 gaps; Z = 58 1 gap; Z = 87 Human pp32r1 AF008216 234 100%Identity 785 Identity 2 61% Identity 90% identity 3 gaps; Z = 65 5 gaps;Z = 15 gaps; Z = 64 Human pp32r2 HSU71084 131 100% Identity 61% Identity77% Identity 3 gaps; Z = 52 1 gap; Z = 80 Human April Y07969 249 100%71% Identity 4 gaps; Z = 68 Murine pp32 U734778 247 100% Identity

[0156] TABLE 3A pp32 Homologs human pp32 (Genbank Locus HSU73477) murinepp32 (Genbank Locus MMU73478) human cerebellar leucine rich acidicnuclear protein (LANP) (Genbank Locus AF025684) murine LANP (GenbankLocus AF022957) murine RFC1 (Genbank Locus MUSMRFC, Accession NO.L23755) HPP2a or human potent heat-stable protein phospatase 2ainhibitor (Genbank Locus HSU60823) SSP29 (Genbank Locus HSU70439) HLA-DRassociated protein I (Genbank Locus HSPPHAPI, Accession No. X75090)PHAPI2a (EMBL Locus HSPHAPI2A, Genbank Accession No. Y07569) PHAPI2b(EMBL Locus HSPHAPI2B, Genbank Accession No. Y07570) April (EMBL LocusHSAPRIL)

[0157] TABLE 6A Tumorigenicity in Nude Mice of NIH3T3 Cells Transfectedwith pp32 and pp32 Variants pp32 Species Clone Tumors/ Average TumorWeight FT1.7 1 3/3 14.9 ± 2.1 2¹ 3/3 13.3 ± 3.7 FT2.2 1 3/3 10.5 ± 2.8 23/3  3.8 ± 2.1 FT2.4 1 3/3⁶  1.3 ± 0.9 2 3/3 13.8 ± 3.3 D3 5² 0/3 6² 0/3P3 11 3/3  5.7 ± 0.5 14³ 3/3  2.1 ± 1.2 P8 1⁴ 3/3  6.4 ± 5.3 2 3/3 11.3± 3.9 4⁵ 3/3 10.1 ± 4.8 L3 (pp32) 5⁵ 0/3 6⁴ 0/3 Vector Control 2³ 0/3 3¹0/3

[0158]

1 51 1 5785 DNA Homo sapiens CDS (4453)..(5154) 1 aagctttcct gatctctaaatcaaggtcag ctccctaagc tcttggctcc cgtactgaaa 60 ctttttctta tgtaactctcataaacacat agcataatgt tttgcatgtt tttcttccct 120 atcagttgca agttccagcagagctgatat attttcattt cattcgctac tatagcccta 180 gagcctgaca tagtttctggctgtgaatgc tcaataaata tttgtttaat tgagtagaaa 240 cataaagtat ctatttcattgaaggaaaga ataattagct acatttttct ttttcttgcc 300 ttaatatttg aggaatttgcttatatgtca taataaaaaa gttaaagcct tatacattat 360 actaaggaat ttggacattaaattcaagct agcctttcta taaacaaaat actgaatttc 420 tgtccctaaa tttgttccttccctattctt ccccattgag atgacaccaa atccctctag 480 ctgctcaaac caagtacccgtatgttattc ttaattatct ctttaccttg cttctcatat 540 gcaatttgtt aacaagtcatcttcagtctg tatccattat tctccctttc cagaccacca 600 acatgtcttg actatactgctacaatagcc tcccaactct tgtcctactt aaaattcatt 660 gtaaaaaatc agtcttggccgggcacggtg gctcacacct ataatcccag cactttggga 720 gtcccaggcg ggcgggtcacgaggtcaaga gatggagacc atcatggcca acatggtgaa 780 accctgtctc tactataaatacaaaaaaat tatctgggtg tggtggcaca tgcctgtaat 840 cccaactact agggaggctgaggcaggaga atcgcttgaa cctgggaggc ggaggttgca 900 gtgagccgag atcgcaccattgcactccag cctggcaaca gagcgagact ccatcccaaa 960 acaaaacaaa acaaaaccatgtaaaacatg tctgtaaaac atgtcagatt tcgtgttcag 1020 aagtcttaca tgtcttttcattatgctaag ataaaaccca aatgcatttt cttggtttct 1080 aaagccaaga aaataagagttgctttcagc aaccttgttt cttccgccat gcttttccct 1140 agctcactct ttttaggcaagtcgacctga ttttctttct gttagtctgt ttctgcctcg 1200 tggtctggct ttctttctgttagtctgttt ccacctcgtg gtcttggtcc tggctcttca 1260 ttctgcctgg aatgctctccactccagatc cttactagat cttagctcag tcatcaccct 1320 cgcaggaaga tcttccaaccattcacctgc atacacctat ggctgctccc tagagaacat 1380 cattctgttt tcttcacttcctagcactta ctgctttctg aaattatcta ctttgattgt 1440 ttatttcttt ctttactcttactaggatac ctgggtcatt aaaggaggga tatttctctc 1500 ttatttactg ttataaacttaatgcttagg ctgtagaagt tatacaatat ttgaagaata 1560 aatcgttaaa tgtataacatttttgaagaa agataattgt gggatccatt tagtttgcaa 1620 acatttgatc tgtgtgttagacagaaggcc atggtaaagg acaaagacat attttatagg 1680 actgtaccct gaaaaataaataaacttgaa ccagttatac aagacttatg tgcaggaaac 1740 aggtaccagt tatatttagaaatggtaaat caccttctaa gcataactca gagcacaata 1800 tattagaggg tagagagagaagtgcgtctt agatattggt aatcatatta ggactgacgc 1860 catccttgat ttttcttctgggaaacagct caaaatgact atttaatgtt tacaatgata 1920 tcttgcatct tgccagtaaataatataata gacactagga atccaaattg taagatgaac 1980 aagtctttat agagggagagccaaatacac aataaataac acaaggtggt aaatgcagta 2040 atacaaacat acataccatgcataggagtg cagagaaggt gtgcttctcc gaatgcagtc 2100 acccagaaag tccttctgtagaaagggata tcttaaatgg tgcttaaagg aaaagtaacc 2160 aaaggcaact aaagattgcaaggaggtccc aggaaaaagc aaaagaacca aaggtacata 2220 ggcacaaaag tagcctgccttcctgggaac ttccaatagt ttgctggagc acacagttag 2280 aagtactgtg ccatgggagcaaagactgaa gacatatgca ggttcaaggg cacagagccc 2340 catatatgtc atgataagatattgggaagc cactggggag ctactgaaac tttaagcagg 2400 gaaataaaat tgtcatatctacaccttaga aatttgattt ttttctcttc ttttatcttc 2460 tcttctcctc tcttctctctctctctctct ctctctctct gtgtgtgtgt gtgtgtgtgt 2520 gtgtgtgtgt gacagagtcctgctctgtca cccaggctgg agtgtagtgg agtgatctcc 2580 gcttactgca gtctctgcctctcaagcgat tccctgcctc agcctcccga gtagctggga 2640 ttacaggcgg gctctacaacagctggctaa cttttgtatt ttttggtaac aaccaggttt 2700 taccatgttg gccaggctggtcttgaactc ctgacctcag gtgatctgcc tgccttggct 2760 ttccaaagtg ctgggattacaggcgtgagc caccctgcct ggtgtagaag tttgattttg 2820 atgtcagtgt ggtagatgaatttgtgggaa gcaaaacaag atagagttca atgacagtga 2880 aaagtttatt gtataagctatataaaagaa aatgttgaag gtttgaaatc cattagtggc 2940 agtaagggtg tacagaacgaaactatttga gaagtacaca aggcaagtct tactttcaag 3000 gcagtttatg taagctcattcaattgtctc agtgttcttg ctatgtgtgg gttataggat 3060 ttggaacata tgatcaatctgagcacacat cagtaaactg aataggatta ttaaaatcca 3120 caagcatttt actagtggaatctgtgatat tttctagcta ctcttgcttg ttttatttga 3180 atcttttgct catatcctatagtaaagatt tcaggaaata tatttttatt tgcctagaat 3240 tttagccttt tagttttttgaatctattgc tcatattctt atagtaagag tttcagggaa 3300 tgtatttcta tttgtctggaattttagcct ttcaggtttt tgagcccctc ttttgcttat 3360 gggacatagt atgagacaagatgaaatgat acttctattc ccaattcact gatggggaaa 3420 atgaagcaaa aaatgttattcactcaaggc ttctgccatg tttcctggtg gaattacggc 3480 tcagacacaa atttcctaatgcctgtgctg ctaacttctc aatagaacac tatattaatt 3540 tatcttcttc ctgagtgtttttccacaaat cccatagcct gtgaaaagat tgttttaggg 3600 aaatattatt tttaatatagcatattttgt caatgtggga cataggacta gtacctgctg 3660 aaaaccatct catgatccttgtgtaagaac taattcacac tagaaatact attttccttg 3720 ctcattaaaa acataaatgtctcagaaagt aaaaaattat tcctctctaa ataaacatac 3780 atgccactca aattttattcctctaccact tgccgtatct aaacctagtt agatactttg 3840 gttttaggta taatctgacagaacagatac aaccaagatc acattgtgag tcagaagtgg 3900 aaaattcata attcatgatgataccaataa aagatagatt tagcttttta caggatgttt 3960 ttggcatttt attctttcatttgaggggag atctcaccaa aatatgtctt tcatggttca 4020 ttgtgttatt taatttctgtgatgcatatt ctcaggttac tttaaaccta gtctatagat 4080 tcaaagatat cccgtgtcaggtctctaaaa gtaaaaagaa aaatgggtac ttgtgaaggc 4140 tgattcacag taagtagtgtagaggggagt gccttgtgta ttcacaaatt atcaacgtga 4200 gcatcagata agattttctttagtcacaca cacctacctt cttactagga agatccatat 4260 acttgaataa ttgttctgcttgacccaggt tacttatcag tccctttatt ataatatttg 4320 taaatattgg ggctcgagaaccgagcggag ctggttgagt cttcaaagtc ctaaaacgtg 4380 cggccgtggg ttcgaggtttattgattgaa ttcggctggc acgagagcct ctgcagacag 4440 agagcgcgag ag atg gagatg ggc aga cgg att cat tca gag ctg cgg aac 4491 Met Glu Met Gly Arg ArgIle His Ser Glu Leu Arg Asn 1 5 10 agg gcg ccc tct gat gtg aaa gaa cttgcc ctg gac aac agt cgg tcg 4539 Arg Ala Pro Ser Asp Val Lys Glu Leu AlaLeu Asp Asn Ser Arg Ser 15 20 25 aat gaa ggc aaa ctc gaa gcc ctc aca gatgaa ttt gaa gaa ctg gaa 4587 Asn Glu Gly Lys Leu Glu Ala Leu Thr Asp GluPhe Glu Glu Leu Glu 30 35 40 45 ttc tta agt aaa atc aac gga ggc ctc acctca atc tca gac tta cca 4635 Phe Leu Ser Lys Ile Asn Gly Gly Leu Thr SerIle Ser Asp Leu Pro 50 55 60 aag tta aag ttg aga aag ctt gaa cta aga gtctca ggg ggc ctg gaa 4683 Lys Leu Lys Leu Arg Lys Leu Glu Leu Arg Val SerGly Gly Leu Glu 65 70 75 gta ttg gca gaa aag tgt cca aac ctc acg cat ctatat tta agt ggc 4731 Val Leu Ala Glu Lys Cys Pro Asn Leu Thr His Leu TyrLeu Ser Gly 80 85 90 aac aaa att aaa gac ctc agc aca ata gag cca ctg aaacag tta gaa 4779 Asn Lys Ile Lys Asp Leu Ser Thr Ile Glu Pro Leu Lys GlnLeu Glu 95 100 105 aac ctc aag agc tta gac ctt ttc aat tgc gag gta accaac ctg aac 4827 Asn Leu Lys Ser Leu Asp Leu Phe Asn Cys Glu Val Thr AsnLeu Asn 110 115 120 125 gac tac gga gaa aac gtg ttc aag ctt ctc ctg caactc aca tat ctc 4875 Asp Tyr Gly Glu Asn Val Phe Lys Leu Leu Leu Gln LeuThr Tyr Leu 130 135 140 gac agc tgt tac tgg gac cac aag gag gcc cct tactca gat att gag 4923 Asp Ser Cys Tyr Trp Asp His Lys Glu Ala Pro Tyr SerAsp Ile Glu 145 150 155 gac cac gtg gag ggc ctg gat gac gag gag gag ggtgag cat gag gag 4971 Asp His Val Glu Gly Leu Asp Asp Glu Glu Glu Gly GluHis Glu Glu 160 165 170 gag tat gat gaa gat gct cag gta gtg gaa gat gaggag ggc gag gag 5019 Glu Tyr Asp Glu Asp Ala Gln Val Val Glu Asp Glu GluGly Glu Glu 175 180 185 gag gag gag gaa ggt gaa gag gag gac gtg agt ggaggg gac gag gag 5067 Glu Glu Glu Glu Gly Glu Glu Glu Asp Val Ser Gly GlyAsp Glu Glu 190 195 200 205 gat gaa gaa ggt tat aac gat gga gag gta gatggc gag gaa gat gaa 5115 Asp Glu Glu Gly Tyr Asn Asp Gly Glu Val Asp GlyGlu Glu Asp Glu 210 215 220 gaa gag ctt ggt gaa gaa gaa agg ggt cag aagcga aaa tgagaacctg 5164 Glu Glu Leu Gly Glu Glu Glu Arg Gly Gln Lys ArgLys 225 230 aagatgaggg agaagatgat gactaagtag aataacctat tttgaaaaattcctattgtg 5224 atttgactgt ttttacccat atcccctccc ccctccaatc ctgccccctgaaacttactt 5284 ttttctgatt gtaacattgc tgtgggaatg agacgggaaa agtgtactgggggttgtgga 5344 gggagggagg gcaggaggcg gtggactaaa atactatttt tactgccaaataaaataata 5404 tttgtaaata ttaactggga tactagcttt gtagaatgat tactattaattattctctct 5464 ctctttttat ttttttacac attctattct tttaagtata gtccttttagtccaaggaaa 5524 aggcactaca atccacttat taatgcttgc tactgtgttc aagtaaaataagctccagga 5584 tttaacaaaa agaggaaaga aaatatttac aatgaaaatg ttgctaaaaatttaaaacaa 5644 attacagtaa atgtattgtt aaagcaaatt ctatttttaa aatttattaataaggaaata 5704 atttgctaaa gcaaattttt ggaaaaataa taatgcactt tatacttgattttatttatt 5764 aaaacaatga tttataagct t 5785 2 234 PRT Homo sapiens 2Met Glu Met Gly Arg Arg Ile His Ser Glu Leu Arg Asn Arg Ala Pro 1 5 1015 Ser Asp Val Lys Glu Leu Ala Leu Asp Asn Ser Arg Ser Asn Glu Gly 20 2530 Lys Leu Glu Ala Leu Thr Asp Glu Phe Glu Glu Leu Glu Phe Leu Ser 35 4045 Lys Ile Asn Gly Gly Leu Thr Ser Ile Ser Asp Leu Pro Lys Leu Lys 50 5560 Leu Arg Lys Leu Glu Leu Arg Val Ser Gly Gly Leu Glu Val Leu Ala 65 7075 80 Glu Lys Cys Pro Asn Leu Thr His Leu Tyr Leu Ser Gly Asn Lys Ile 8590 95 Lys Asp Leu Ser Thr Ile Glu Pro Leu Lys Gln Leu Glu Asn Leu Lys100 105 110 Ser Leu Asp Leu Phe Asn Cys Glu Val Thr Asn Leu Asn Asp TyrGly 115 120 125 Glu Asn Val Phe Lys Leu Leu Leu Gln Leu Thr Tyr Leu AspSer Cys 130 135 140 Tyr Trp Asp His Lys Glu Ala Pro Tyr Ser Asp Ile GluAsp His Val 145 150 155 160 Glu Gly Leu Asp Asp Glu Glu Glu Gly Glu HisGlu Glu Glu Tyr Asp 165 170 175 Glu Asp Ala Gln Val Val Glu Asp Glu GluGly Glu Glu Glu Glu Glu 180 185 190 Glu Gly Glu Glu Glu Asp Val Ser GlyGly Asp Glu Glu Asp Glu Glu 195 200 205 Gly Tyr Asn Asp Gly Glu Val AspGly Glu Glu Asp Glu Glu Glu Leu 210 215 220 Gly Glu Glu Glu Arg Gly GlnLys Arg Lys 225 230 3 889 DNA Homo sapiens 3 gggttcgagg tttattgattgaattcggct ggcacgagag cctctgcaga cagagagcgc 60 gagagatgga gatgggcagacggattcatt cagagctgcg gaacagggcg ccctctgatg 120 tgaaagaact tgccctggacaacagtcggt cgaatgaagg caaactcgaa gccctcacag 180 atgaatttga agaactggaattcttaagta aaatcaacgg aggcctcacc tcaatctcag 240 acttaccaaa gttaaagttgagaaagcttg aactaagagt ctcagggggc ctggaagtat 300 tggcagaaaa gtgtccaaacctcacgcatc tatatttaag tggcaacaaa attaaagacc 360 tcagcacaat agagccactgaaacagttag aaaacctcaa gagcttagac cttttcaatt 420 gcgaggtaac caacctgaacgactacggag aaaacgtgtt caagcttctc ctgcaactca 480 catatctcga cagctgttactgggaccaca aggaggcccc ttactcagat attgaggacc 540 acgtggaggg cctggatgacgaggaggagg gtgagcatga ggaggagtat gatgaagatg 600 ctcaggtagt ggaagatgaggagggcgagg aggaggagga ggaaggtgaa gaggaggacg 660 tgagtggagg ggacgaggaggatgaagaag gttataacga tggagaggta gatggcgagg 720 aagatgaaga agagcttggtgaagaagaaa ggggtcagaa gcgaaaatga gaacctgaag 780 atgagggaga agatgatgactaagtagaat aacctatttt gaaaaattcc tattgtgatt 840 tgactgtttt tacccatatcccctcccccc tccaatcctg ccccctgaa 889 4 907 DNA Homo sapiens 4 gggttcggggtttattgatt gaattcggct ggcgcgggag cctctgcaga gagagagcgc 60 gagagatggagatgggcaga cggattcatt tagagctgcg gaacgggacg ccctctgatg 120 tgaaagaacttgtcctggac aacagtcggt cgaatgaagg caaactcgaa ggcctcacag 180 atgaatttgaagaactggaa ttcttaagta caatcaacgt aggcctcacc tcaatcgcaa 240 acttaccaaagttaaacaaa cttaagaagc ttgaactaag cagtaacaga gcctcagtgg 300 gcctagaagtattggcagaa aagtgtccaa acctcataca tctaaattta agtggcaaca 360 aaattaaagacctcagcaca atagagcccc tgaaaaagtt agaaaacctc gagagcttag 420 accttttcacttgcgaggta accaacctga acaactactg agagaagatg ttcaagctcc 480 tcctgcaactcacatatctc aacggctgtg acccggatga caaggaggcc cctaactcgg 540 atggtgagggctttgtggag tgcctggatg acaaggagga ggatgaggat gaggaggagt 600 atgatgaagatgctcaggta atggaagatg aggaggacga ggatgaggag gaggaacgtg 660 aagaggaggacgtgagtgga gacgaggagg agaaggatga aggttataac aatggagagg 720 tagatgatgaggaagatgaa gaagagcttg gtgaagaaga aaggggtcag aagcgaaaat 780 aagaaactgaagatgaggga gaagacgatg cctaagtgga ataatctatt ttgaaaaatt 840 ccttttgtgattttactgtt tttagccgta ccccctctcc ccccccactc taatcctgcc 900 ccctgaa 907 5130 PRT Homo sapiens 5 Met Glu Met Gly Arg Arg Ile His Leu Glu Leu ArgAsn Gly Thr Pro 1 5 10 15 Ser Asp Val Lys Glu Leu Val Leu Asp Asn SerArg Ser Asn Glu Gly 20 25 30 Lys Leu Glu Gly Leu Thr Asp Glu Phe Glu GluLeu Glu Phe Leu Ser 35 40 45 Thr Ile Asn Val Gly Leu Thr Ser Ile Ala AsnLeu Pro Lys Leu Asn 50 55 60 Lys Leu Lys Lys Leu Glu Leu Ser Ser Asn ArgAla Ser Val Gly Leu 65 70 75 80 Glu Val Leu Ala Glu Lys Cys Pro Asn LeuIle His Leu Asn Leu Ser 85 90 95 Gly Asn Lys Ile Lys Asp Leu Ser Thr IleGlu Pro Leu Lys Lys Leu 100 105 110 Glu Asn Leu Glu Ser Leu Asp Leu PheThr Cys Glu Val Thr Asn Leu 115 120 125 Asn Asn 130 6 907 DNA Homosapiens 6 gggttcgggg tttattgatt gaattcggct ggcacgagag cctctgcagacagagagcgc 60 gagagacgga gatgggcaga cggattcatc tagagctgcg gaacagggcgccctctgatg 120 tgaaagaact tgccctggac aacagtcggt cgaatgaagg caaactcgaagccctcacag 180 atgaatttga agaactggaa ttcttaagta aaatcaacgg aggcctcacctcaatctcag 240 acttaccaaa gttaaacaag ttgagaaagc ttgaactaag cagtaacagagtctcagggg 300 gcctggaagt attggcagaa aagtgtccaa acctcacgca tctatatttaagtggcaaca 360 aaattaaaga cctcagcaca atagagccac tgaaacagtt agaaaacctcaagagcttag 420 accttttcaa ttgcgaggta accaacctga acgactacgg agaaaacgtgttcaagcttc 480 tcctgcaact cacatatctc gacagctgtt actgggacca caaggaggccccttactcag 540 atattgaggc ccacgtggag ggcctggatg acgaggagga gggtgagcatgaggaggagt 600 atgatgaaga tgctcaggta gtggaagatg aggagggcga ggaggaggaggaggaaggtg 660 aagaggagga cgtgagtgga ggggacgagg aggatgaaga aggttataacgatggagagg 720 tagatggcga ggaagatgaa gaagagcttg gtgaagaaga aaggggtcagaagcgaaaat 780 gagaacctga agatgaggga gaagatgatg actaagtaga ataacctattttgaaaaatt 840 cctattgtga tttgactgtt tttacccata tcccctctcc cccccccctctaatcctgcc 900 ccctgaa 907 7 905 DNA Homo sapiens CDS (64)..(453) 7gggttcgggg tttattggtt gaattccgct ggctcaggag cctctgcaga gaaagcgtga 60 gagatg gag atg ggc aaa tgg att cat tta gag ctg cgg aac agg acg 108 Met GluMet Gly Lys Trp Ile His Leu Glu Leu Arg Asn Arg Thr 1 5 10 15 ccc tccgat gtg aaa gaa ctt ttc ctg gac aac agt cag tca aat gaa 156 Pro Ser AspVal Lys Glu Leu Phe Leu Asp Asn Ser Gln Ser Asn Glu 20 25 30 ggc aaa ttggaa ggc ctc aca gat gaa ttt gaa gaa ctg gaa tta tta 204 Gly Lys Leu GluGly Leu Thr Asp Glu Phe Glu Glu Leu Glu Leu Leu 35 40 45 aat aca atc aacata ggc ctc acc tca att gca aac ttg cca aag tta 252 Asn Thr Ile Asn IleGly Leu Thr Ser Ile Ala Asn Leu Pro Lys Leu 50 55 60 aac aaa ctt aag aagctt gaa cta agc agt aac aga gcc tca gtg ggc 300 Asn Lys Leu Lys Lys LeuGlu Leu Ser Ser Asn Arg Ala Ser Val Gly 65 70 75 cta gaa gta ttg gca gaaaag tgt cca aac ctc ata cat cta aat tta 348 Leu Glu Val Leu Ala Glu LysCys Pro Asn Leu Ile His Leu Asn Leu 80 85 90 95 agt ggc aac aaa att aaagac ctc agc aca ata gag ccc ctg aaa aag 396 Ser Gly Asn Lys Ile Lys AspLeu Ser Thr Ile Glu Pro Leu Lys Lys 100 105 110 tta gaa aac ctc gag agctta gac ctt ttc act tgc gag gta acc aac 444 Leu Glu Asn Leu Glu Ser LeuAsp Leu Phe Thr Cys Glu Val Thr Asn 115 120 125 ctg aac aac tactgagaaaagatgttcaa gctcctcctg caactcacat 493 Leu Asn Asn 130 atctcaacggctgtgacccg gatgacaagg aggcccctaa ctcggatggt gagggctatg 553 tggagtgcctggatgacaag gaggaggatg aggatgagga ggagtatgat gaagatgctc 613 aggtaatggaagatgaggag gacgaggatg aggaggagga acgtgaagag gaggacgtga 673 gtggagacgaggaggagaag gatgaaggtt ataacaatgg agaggtagat gatgaggaag 733 atgaagaagagcttggtgaa gaagaaaggg gtcagaagcg aaaataagaa actgaagatg 793 agggagaagacgatgcctaa gtggaataat ctattttgaa aaattccttt tgtgatttta 853 ctgtttttagccgtatcccc tctccccccc cactctaatc ctgccccctg aa 905 8 130 PRT Homosapiens 8 Met Glu Met Gly Lys Trp Ile His Leu Glu Leu Arg Asn Arg ThrPro 1 5 10 15 Ser Asp Val Lys Glu Leu Phe Leu Asp Asn Ser Gln Ser AsnGlu Gly 20 25 30 Lys Leu Glu Gly Leu Thr Asp Glu Phe Glu Glu Leu Glu LeuLeu Asn 35 40 45 Thr Ile Asn Ile Gly Leu Thr Ser Ile Ala Asn Leu Pro LysLeu Asn 50 55 60 Lys Leu Lys Lys Leu Glu Leu Ser Ser Asn Arg Ala Ser ValGly Leu 65 70 75 80 Glu Val Leu Ala Glu Lys Cys Pro Asn Leu Ile His LeuAsn Leu Ser 85 90 95 Gly Asn Lys Ile Lys Asp Leu Ser Thr Ile Glu Pro LeuLys Lys Leu 100 105 110 Glu Asn Leu Glu Ser Leu Asp Leu Phe Thr Cys GluVal Thr Asn Leu 115 120 125 Asn Asn 130 9 907 DNA Homo sapiens CDS(66)..(812) 9 gggttcgggg tttattgatt gaattccgcc ggcgcgggag cctctgcagagagagagcgc 60 gagag atg gag atg ggc aga cgg att cat tta gag ctg cgg aacagg acg 110 Met Glu Met Gly Arg Arg Ile His Leu Glu Leu Arg Asn Arg Thr1 5 10 15 ccc tct gat gtg aaa gaa ctt gtc ctg gac aac agt cgg tcg aatgaa 158 Pro Ser Asp Val Lys Glu Leu Val Leu Asp Asn Ser Arg Ser Asn Glu20 25 30 ggc aaa ctc gaa ggc ctc aca gat gaa ttt gaa gaa ctg gaa ttc tta206 Gly Lys Leu Glu Gly Leu Thr Asp Glu Phe Glu Glu Leu Glu Phe Leu 3540 45 agt aca atc aac gta ggc ctc acc tca atc gca aac ttg cca aag tta254 Ser Thr Ile Asn Val Gly Leu Thr Ser Ile Ala Asn Leu Pro Lys Leu 5055 60 aac aaa ctt aag aag ctt gaa cta agc agt aac aga gcc tca gtg ggc302 Asn Lys Leu Lys Lys Leu Glu Leu Ser Ser Asn Arg Ala Ser Val Gly 6570 75 cta gaa gta ttg gca gaa aag tgt cca aac ctc ata cat cta aat tta350 Leu Glu Val Leu Ala Glu Lys Cys Pro Asn Leu Ile His Leu Asn Leu 8085 90 95 agt ggc aac aaa att aaa gac ctc agc aca ata gag cca ctg aaa aag398 Ser Gly Asn Lys Ile Lys Asp Leu Ser Thr Ile Glu Pro Leu Lys Lys 100105 110 tta gaa aac ctc aag agc tta gac ctt tcc aat tgc gag gta acc aac446 Leu Glu Asn Leu Lys Ser Leu Asp Leu Ser Asn Cys Glu Val Thr Asn 115120 125 ctg aac gac tac cga gaa aat gtg ttc aag ctc ctc ccg caa ctc aca494 Leu Asn Asp Tyr Arg Glu Asn Val Phe Lys Leu Leu Pro Gln Leu Thr 130135 140 tat ctc gac ggc tat gac cgg gac gac aag gag gcc cct gac tcg gat542 Tyr Leu Asp Gly Tyr Asp Arg Asp Asp Lys Glu Ala Pro Asp Ser Asp 145150 155 gct gag ggc tac gtg gag ggc ctg gat gat gag gag gag gat gag gat590 Ala Glu Gly Tyr Val Glu Gly Leu Asp Asp Glu Glu Glu Asp Glu Asp 160165 170 175 gag gag gag tat gat gaa gat gct cag gta gta gaa gat gag gaggac 638 Glu Glu Glu Tyr Asp Glu Asp Ala Gln Val Val Glu Asp Glu Glu Asp180 185 190 gag gat gag gag gag gaa ggt gaa gag gag gac gtg agt gga gaggag 686 Glu Asp Glu Glu Glu Glu Gly Glu Glu Glu Asp Val Ser Gly Glu Glu195 200 205 gag gag gat gaa gaa ggt tat aac gat gga gag gta gat gac gaggaa 734 Glu Glu Asp Glu Glu Gly Tyr Asn Asp Gly Glu Val Asp Asp Glu Glu210 215 220 gat gaa gaa gag ctt ggt gaa gaa gaa agg ggt cag aag cga aaacga 782 Asp Glu Glu Glu Leu Gly Glu Glu Glu Arg Gly Gln Lys Arg Lys Arg225 230 235 gaa cct gaa gat gag gga gaa gat gat gac taagtggaataacctatttt 832 Glu Pro Glu Asp Glu Gly Glu Asp Asp Asp 240 245gaaaaattcc tattgtgatt tgactgtttt tacccatatc ccctctcccc cccccctcta 892atcctgcccc ctgaa 907 10 249 PRT Homo sapiens 10 Met Glu Met Gly Arg ArgIle His Leu Glu Leu Arg Asn Arg Thr Pro 1 5 10 15 Ser Asp Val Lys GluLeu Val Leu Asp Asn Ser Arg Ser Asn Glu Gly 20 25 30 Lys Leu Glu Gly LeuThr Asp Glu Phe Glu Glu Leu Glu Phe Leu Ser 35 40 45 Thr Ile Asn Val GlyLeu Thr Ser Ile Ala Asn Leu Pro Lys Leu Asn 50 55 60 Lys Leu Lys Lys LeuGlu Leu Ser Ser Asn Arg Ala Ser Val Gly Leu 65 70 75 80 Glu Val Leu AlaGlu Lys Cys Pro Asn Leu Ile His Leu Asn Leu Ser 85 90 95 Gly Asn Lys IleLys Asp Leu Ser Thr Ile Glu Pro Leu Lys Lys Leu 100 105 110 Glu Asn LeuLys Ser Leu Asp Leu Ser Asn Cys Glu Val Thr Asn Leu 115 120 125 Asn AspTyr Arg Glu Asn Val Phe Lys Leu Leu Pro Gln Leu Thr Tyr 130 135 140 LeuAsp Gly Tyr Asp Arg Asp Asp Lys Glu Ala Pro Asp Ser Asp Ala 145 150 155160 Glu Gly Tyr Val Glu Gly Leu Asp Asp Glu Glu Glu Asp Glu Asp Glu 165170 175 Glu Glu Tyr Asp Glu Asp Ala Gln Val Val Glu Asp Glu Glu Asp Glu180 185 190 Asp Glu Glu Glu Glu Gly Glu Glu Glu Asp Val Ser Gly Glu GluGlu 195 200 205 Glu Asp Glu Glu Gly Tyr Asn Asp Gly Glu Val Asp Asp GluGlu Asp 210 215 220 Glu Glu Glu Leu Gly Glu Glu Glu Arg Gly Gln Lys ArgLys Arg Glu 225 230 235 240 Pro Glu Asp Glu Gly Glu Asp Asp Asp 245 11905 DNA Homo sapiens CDS (64)..(810) 11 gggttcgggg tttattggtt gaattccgctggctcaggag cctctgcaga gaaagcgtga 60 gag atg gag atg ggc aaa tgg att cattta gag ctg cgg aac agg acg 108 Met Glu Met Gly Lys Trp Ile His Leu GluLeu Arg Asn Arg Thr 1 5 10 15 ccc tcc gat gtg aaa gaa ctt ttc ctg gacaac agt cag tca aat gaa 156 Pro Ser Asp Val Lys Glu Leu Phe Leu Asp AsnSer Gln Ser Asn Glu 20 25 30 ggc aaa ttg gaa ggc ctc aca gat gaa ttt gaagaa ctg gaa tta tta 204 Gly Lys Leu Glu Gly Leu Thr Asp Glu Phe Glu GluLeu Glu Leu Leu 35 40 45 aat aca atc aac ata ggc ctc acc tca att gca aacttg cca aag tta 252 Asn Thr Ile Asn Ile Gly Leu Thr Ser Ile Ala Asn LeuPro Lys Leu 50 55 60 aac aaa ctt aag aag ctt gaa cta agc agt aac aga gcctca gtg ggc 300 Asn Lys Leu Lys Lys Leu Glu Leu Ser Ser Asn Arg Ala SerVal Gly 65 70 75 cta gaa gta ttg gca gaa aag tgt cca aac ctc ata cat ctaaat tta 348 Leu Glu Val Leu Ala Glu Lys Cys Pro Asn Leu Ile His Leu AsnLeu 80 85 90 95 agt ggc aac aaa att aaa gac ctc agc aca ata gag ccc ctgaaa aag 396 Ser Gly Asn Lys Ile Lys Asp Leu Ser Thr Ile Glu Pro Leu LysLys 100 105 110 tta gaa aac ctc gag agc tta gac ctt ttc act tgc gag gtaacc aac 444 Leu Glu Asn Leu Glu Ser Leu Asp Leu Phe Thr Cys Glu Val ThrAsn 115 120 125 ctg aac aac tac cga gaa aat gtg ttc aag ctc ctc ccg caactc aca 492 Leu Asn Asn Tyr Arg Glu Asn Val Phe Lys Leu Leu Pro Gln LeuThr 130 135 140 tat ctc gac ggc tat gac cgg gac gac aag gag gcc cct gactcg gat 540 Tyr Leu Asp Gly Tyr Asp Arg Asp Asp Lys Glu Ala Pro Asp SerAsp 145 150 155 gct gag ggc tac gtg gag ggc ctg gat gat gag gag gag gatgag gat 588 Ala Glu Gly Tyr Val Glu Gly Leu Asp Asp Glu Glu Glu Asp GluAsp 160 165 170 175 gag gag gag tat gat gaa gat gct cag gta gtg gaa gacgag gag gac 636 Glu Glu Glu Tyr Asp Glu Asp Ala Gln Val Val Glu Asp GluGlu Asp 180 185 190 gag gat gag gag gag gaa ggt gaa gag gag gac gtg agtgga gag gag 684 Glu Asp Glu Glu Glu Glu Gly Glu Glu Glu Asp Val Ser GlyGlu Glu 195 200 205 gag gag gat gaa gaa ggt tat aac gat gga gag gta gatgac gag gaa 732 Glu Glu Asp Glu Glu Gly Tyr Asn Asp Gly Glu Val Asp AspGlu Glu 210 215 220 gat gaa gaa gag ctt ggt gaa gaa gaa agg ggt cag aagcga aaa cga 780 Asp Glu Glu Glu Leu Gly Glu Glu Glu Arg Gly Gln Lys ArgLys Arg 225 230 235 gaa cct gaa gat gag gga gaa gat gat gac taagtggaataacctatttt 830 Glu Pro Glu Asp Glu Gly Glu Asp Asp Asp 240 245gaaaaattcc tattgtgatt tgactgtttt tacccatatc ccctctcccc cccccctcta 890atcctgcccc ctgaa 905 12 249 PRT Homo sapiens 12 Met Glu Met Gly Lys TrpIle His Leu Glu Leu Arg Asn Arg Thr Pro 1 5 10 15 Ser Asp Val Lys GluLeu Phe Leu Asp Asn Ser Gln Ser Asn Glu Gly 20 25 30 Lys Leu Glu Gly LeuThr Asp Glu Phe Glu Glu Leu Glu Leu Leu Asn 35 40 45 Thr Ile Asn Ile GlyLeu Thr Ser Ile Ala Asn Leu Pro Lys Leu Asn 50 55 60 Lys Leu Lys Lys LeuGlu Leu Ser Ser Asn Arg Ala Ser Val Gly Leu 65 70 75 80 Glu Val Leu AlaGlu Lys Cys Pro Asn Leu Ile His Leu Asn Leu Ser 85 90 95 Gly Asn Lys IleLys Asp Leu Ser Thr Ile Glu Pro Leu Lys Lys Leu 100 105 110 Glu Asn LeuGlu Ser Leu Asp Leu Phe Thr Cys Glu Val Thr Asn Leu 115 120 125 Asn AsnTyr Arg Glu Asn Val Phe Lys Leu Leu Pro Gln Leu Thr Tyr 130 135 140 LeuAsp Gly Tyr Asp Arg Asp Asp Lys Glu Ala Pro Asp Ser Asp Ala 145 150 155160 Glu Gly Tyr Val Glu Gly Leu Asp Asp Glu Glu Glu Asp Glu Asp Glu 165170 175 Glu Glu Tyr Asp Glu Asp Ala Gln Val Val Glu Asp Glu Glu Asp Glu180 185 190 Asp Glu Glu Glu Glu Gly Glu Glu Glu Asp Val Ser Gly Glu GluGlu 195 200 205 Glu Asp Glu Glu Gly Tyr Asn Asp Gly Glu Val Asp Asp GluGlu Asp 210 215 220 Glu Glu Glu Leu Gly Glu Glu Glu Arg Gly Gln Lys ArgLys Arg Glu 225 230 235 240 Pro Glu Asp Glu Gly Glu Asp Asp Asp 245 13907 DNA Homo sapiens CDS (66)..(812) 13 gggttcgggg tttattgatt gaattccgccggcgcgggag cctctgcaga gagagagcgc 60 gagag atg gag atg ggc aga cgg attcat tta gag ctg cgg aac agg acg 110 Met Glu Met Gly Arg Arg Ile His LeuGlu Leu Arg Asn Arg Thr 1 5 10 15 ccc tct gat gtg aaa gaa ctt gtc ctggac aac agt cgg tcg aat gaa 158 Pro Ser Asp Val Lys Glu Leu Val Leu AspAsn Ser Arg Ser Asn Glu 20 25 30 ggc aaa ctc gaa ggc ctc aca gat gaa tttgaa gaa ctg gaa ttc tta 206 Gly Lys Leu Glu Gly Leu Thr Asp Glu Phe GluGlu Leu Glu Phe Leu 35 40 45 agt aca atc aac gta ggc ctc acc tca atc gcaaac tta cca aag tta 254 Ser Thr Ile Asn Val Gly Leu Thr Ser Ile Ala AsnLeu Pro Lys Leu 50 55 60 aac aaa ctt aag aag ctt gaa cta agc gat aac agagtc tca ggg ggc 302 Asn Lys Leu Lys Lys Leu Glu Leu Ser Asp Asn Arg ValSer Gly Gly 65 70 75 ctg gaa gta ttg gca gaa aag tgt ccg aac ctc acg catcta aat tta 350 Leu Glu Val Leu Ala Glu Lys Cys Pro Asn Leu Thr His LeuAsn Leu 80 85 90 95 agt ggc aac aaa att aaa gac ctc agc aca ata gag ccactg aaa aag 398 Ser Gly Asn Lys Ile Lys Asp Leu Ser Thr Ile Glu Pro LeuLys Lys 100 105 110 tta gaa aac ctc aag agc tta gac ctt ttc aat tgc gaggta acc aac 446 Leu Glu Asn Leu Lys Ser Leu Asp Leu Phe Asn Cys Glu ValThr Asn 115 120 125 ctg aac gac tac cga gaa aat gtg ttc aag ctc ctc ccgcaa ctc aca 494 Leu Asn Asp Tyr Arg Glu Asn Val Phe Lys Leu Leu Pro GlnLeu Thr 130 135 140 tat ctc gac ggc tat gac cgg gac gac aag gag gcc cctgac tcg gat 542 Tyr Leu Asp Gly Tyr Asp Arg Asp Asp Lys Glu Ala Pro AspSer Asp 145 150 155 gct gag ggc tac gtg gag ggc ctg gat gat gag gag gaggat gag gat 590 Ala Glu Gly Tyr Val Glu Gly Leu Asp Asp Glu Glu Glu AspGlu Asp 160 165 170 175 gag gag gag tat gat gaa gat gct cag gta gtg gaagac gag gag gac 638 Glu Glu Glu Tyr Asp Glu Asp Ala Gln Val Val Glu AspGlu Glu Asp 180 185 190 gag gat gag gag gag gaa ggt gaa gag gag gac gtgagt gga gag gag 686 Glu Asp Glu Glu Glu Glu Gly Glu Glu Glu Asp Val SerGly Glu Glu 195 200 205 gag gag gat gaa gaa ggt tat aac gat gga gag gtagat gac gag gaa 734 Glu Glu Asp Glu Glu Gly Tyr Asn Asp Gly Glu Val AspAsp Glu Glu 210 215 220 gat gaa gaa gag ctt ggt gaa gaa gaa agg ggt cagaag cga aaa cga 782 Asp Glu Glu Glu Leu Gly Glu Glu Glu Arg Gly Gln LysArg Lys Arg 225 230 235 gaa cct gaa gat gag gga gaa gat gat gactaagtggaat aacctatttt 832 Glu Pro Glu Asp Glu Gly Glu Asp Asp Asp 240245 gaaaaattcc tattgtgatt tgactgtttt tacccatatc ccctctcccc cccccctcta892 atcctgcccc ctgaa 907 14 249 PRT Homo sapiens 14 Met Glu Met Gly ArgArg Ile His Leu Glu Leu Arg Asn Arg Thr Pro 1 5 10 15 Ser Asp Val LysGlu Leu Val Leu Asp Asn Ser Arg Ser Asn Glu Gly 20 25 30 Lys Leu Glu GlyLeu Thr Asp Glu Phe Glu Glu Leu Glu Phe Leu Ser 35 40 45 Thr Ile Asn ValGly Leu Thr Ser Ile Ala Asn Leu Pro Lys Leu Asn 50 55 60 Lys Leu Lys LysLeu Glu Leu Ser Asp Asn Arg Val Ser Gly Gly Leu 65 70 75 80 Glu Val LeuAla Glu Lys Cys Pro Asn Leu Thr His Leu Asn Leu Ser 85 90 95 Gly Asn LysIle Lys Asp Leu Ser Thr Ile Glu Pro Leu Lys Lys Leu 100 105 110 Glu AsnLeu Lys Ser Leu Asp Leu Phe Asn Cys Glu Val Thr Asn Leu 115 120 125 AsnAsp Tyr Arg Glu Asn Val Phe Lys Leu Leu Pro Gln Leu Thr Tyr 130 135 140Leu Asp Gly Tyr Asp Arg Asp Asp Lys Glu Ala Pro Asp Ser Asp Ala 145 150155 160 Glu Gly Tyr Val Glu Gly Leu Asp Asp Glu Glu Glu Asp Glu Asp Glu165 170 175 Glu Glu Tyr Asp Glu Asp Ala Gln Val Val Glu Asp Glu Glu AspGlu 180 185 190 Asp Glu Glu Glu Glu Gly Glu Glu Glu Asp Val Ser Gly GluGlu Glu 195 200 205 Glu Asp Glu Glu Gly Tyr Asn Asp Gly Glu Val Asp AspGlu Glu Asp 210 215 220 Glu Glu Glu Leu Gly Glu Glu Glu Arg Gly Gln LysArg Lys Arg Glu 225 230 235 240 Pro Glu Asp Glu Gly Glu Asp Asp Asp 24515 895 DNA Homo sapiens CDS (66)..(767) 15 gggttcgggg tttattgattgaattcggct ggcacgagag cctctgcaga cagagagcgc 60 gagag atg gag atg ggc agacgg att cat tca gag ctg cgg aac agg gcg 110 Met Glu Met Gly Arg Arg IleHis Ser Glu Leu Arg Asn Arg Ala 1 5 10 15 ccc tct gat gtg aaa gaa cttgcc ctg gac aac agt cgg tcg aat gaa 158 Pro Ser Asp Val Lys Glu Leu AlaLeu Asp Asn Ser Arg Ser Asn Glu 20 25 30 ggc aaa ctc gaa gcc ctc aca gatgaa ttt gaa gaa ctg gaa ttc tta 206 Gly Lys Leu Glu Ala Leu Thr Asp GluPhe Glu Glu Leu Glu Phe Leu 35 40 45 agt aaa atc aac gga ggc ctc acc tcaatc tca gac tta cca aag tta 254 Ser Lys Ile Asn Gly Gly Leu Thr Ser IleSer Asp Leu Pro Lys Leu 50 55 60 aag ttg aga aag ctt gaa cta aga gtc tcaggg ggc ctg gaa gta ttg 302 Lys Leu Arg Lys Leu Glu Leu Arg Val Ser GlyGly Leu Glu Val Leu 65 70 75 gca gaa aag tgt cca aac ctc acg cat cta tattta agt ggc aac aaa 350 Ala Glu Lys Cys Pro Asn Leu Thr His Leu Tyr LeuSer Gly Asn Lys 80 85 90 95 att aaa gac ctc agc aca ata gag cca ctg aaacag tta gaa aac ctc 398 Ile Lys Asp Leu Ser Thr Ile Glu Pro Leu Lys GlnLeu Glu Asn Leu 100 105 110 aag agc tta gac ctt ttc aat tgc gag gta accaac ctg aac gac tac 446 Lys Ser Leu Asp Leu Phe Asn Cys Glu Val Thr AsnLeu Asn Asp Tyr 115 120 125 gga gaa aac gtg ttc aag ctt ctc ctg caa ctcaca tat ctc gac agc 494 Gly Glu Asn Val Phe Lys Leu Leu Leu Gln Leu ThrTyr Leu Asp Ser 130 135 140 tgt tac tgg gac cac aag gag gcc cct tac tcagat att gag gac cac 542 Cys Tyr Trp Asp His Lys Glu Ala Pro Tyr Ser AspIle Glu Asp His 145 150 155 gtg gag ggc ctg gat gac gag gag gag ggt gagcat gag gag gag tat 590 Val Glu Gly Leu Asp Asp Glu Glu Glu Gly Glu HisGlu Glu Glu Tyr 160 165 170 175 gat gaa gat gct cag gta gtg gaa gat gaggag ggc gag gag gag gag 638 Asp Glu Asp Ala Gln Val Val Glu Asp Glu GluGly Glu Glu Glu Glu 180 185 190 gag gaa ggt gaa gag gag gac gtg agt ggaggg gac ggg gag gat gaa 686 Glu Glu Gly Glu Glu Glu Asp Val Ser Gly GlyAsp Gly Glu Asp Glu 195 200 205 gaa ggt tat aac gat gga gag gta gat ggcgag gaa gat gaa gaa gag 734 Glu Gly Tyr Asn Asp Gly Glu Val Asp Gly GluGlu Asp Glu Glu Glu 210 215 220 ctt ggt gaa gaa gaa agg ggt cag aag cgaaaa tgagaacctg aagatgaggg 787 Leu Gly Glu Glu Glu Arg Gly Gln Lys ArgLys 225 230 agaagatgat gactaagtag aataacctat tttgaaaaat tcctattgtgatttgactgt 847 ttttacccat atcccatctc ccccccccct ctaatcctgc cccctgaa 89516 234 PRT Homo sapiens 16 Met Glu Met Gly Arg Arg Ile His Ser Glu LeuArg Asn Arg Ala Pro 1 5 10 15 Ser Asp Val Lys Glu Leu Ala Leu Asp AsnSer Arg Ser Asn Glu Gly 20 25 30 Lys Leu Glu Ala Leu Thr Asp Glu Phe GluGlu Leu Glu Phe Leu Ser 35 40 45 Lys Ile Asn Gly Gly Leu Thr Ser Ile SerAsp Leu Pro Lys Leu Lys 50 55 60 Leu Arg Lys Leu Glu Leu Arg Val Ser GlyGly Leu Glu Val Leu Ala 65 70 75 80 Glu Lys Cys Pro Asn Leu Thr His LeuTyr Leu Ser Gly Asn Lys Ile 85 90 95 Lys Asp Leu Ser Thr Ile Glu Pro LeuLys Gln Leu Glu Asn Leu Lys 100 105 110 Ser Leu Asp Leu Phe Asn Cys GluVal Thr Asn Leu Asn Asp Tyr Gly 115 120 125 Glu Asn Val Phe Lys Leu LeuLeu Gln Leu Thr Tyr Leu Asp Ser Cys 130 135 140 Tyr Trp Asp His Lys GluAla Pro Tyr Ser Asp Ile Glu Asp His Val 145 150 155 160 Glu Gly Leu AspAsp Glu Glu Glu Gly Glu His Glu Glu Glu Tyr Asp 165 170 175 Glu Asp AlaGln Val Val Glu Asp Glu Glu Gly Glu Glu Glu Glu Glu 180 185 190 Glu GlyGlu Glu Glu Asp Val Ser Gly Gly Asp Gly Glu Asp Glu Glu 195 200 205 GlyTyr Asn Asp Gly Glu Val Asp Gly Glu Glu Asp Glu Glu Glu Leu 210 215 220Gly Glu Glu Glu Arg Gly Gln Lys Arg Lys 225 230 17 905 DNA Homo sapiensCDS (64)..(453) 17 gggttcgggg tttattggtt gaattccgct ggctcgagagcctctggaga gaaagcgtga 60 gag atg gag atg ggc aaa tgg att cat tta gag ctgcgg aac agg acg 108 Met Glu Met Gly Lys Trp Ile His Leu Glu Leu Arg AsnArg Thr 1 5 10 15 ccc tcc gat gtg aaa gaa ctt ttc ctg gac aac agt cagtca aat gaa 156 Pro Ser Asp Val Lys Glu Leu Phe Leu Asp Asn Ser Gln SerAsn Glu 20 25 30 ggc aaa ttg gaa ggc ctc aca gat gaa ttt gag gaa ctg gaatta tta 204 Gly Lys Leu Glu Gly Leu Thr Asp Glu Phe Glu Glu Leu Glu LeuLeu 35 40 45 aat aca atc aac ata ggc ctc acc tca att gca aac ttg cca aagtta 252 Asn Thr Ile Asn Ile Gly Leu Thr Ser Ile Ala Asn Leu Pro Lys Leu50 55 60 aac aaa ctt aag aag ctt gaa cta agc agt aac aga gcc tca gtg ggc300 Asn Lys Leu Lys Lys Leu Glu Leu Ser Ser Asn Arg Ala Ser Val Gly 6570 75 cta gaa gta ttg gca gaa aag tgt cca aac ctc ata cat cta aat tta348 Leu Glu Val Leu Ala Glu Lys Cys Pro Asn Leu Ile His Leu Asn Leu 8085 90 95 agt ggc aac aaa att aaa gac ctc agc aca ata gag ccc ctg aaa aag396 Ser Gly Asn Lys Ile Lys Asp Leu Ser Thr Ile Glu Pro Leu Lys Lys 100105 110 tta gaa aac ctt gag agc tta gac ctt ttc act tgc gag gta acc aac444 Leu Glu Asn Leu Glu Ser Leu Asp Leu Phe Thr Cys Glu Val Thr Asn 115120 125 ctg aac aac tactgagaaa agatgttcaa gctcctcctg caactcacat 493 LeuAsn Asn 130 atctcaacgg ctgtgacccg gatgacaagg aggcccctaa ctcggatggtgagggctacg 553 tggagggcct ggacgatgag gaggaggatg aggatgagga ggagtatgatgaagatgctc 613 aggtagtgga agacgaggag gacgaggatg aggaggagga aggtgaagaggaggacgtga 673 gtggagagga ggaggaggat gaagaaggtt ataacgatgg agaggtagatgacgaggaag 733 atgaagaaga gcttggtgaa gaagaaaggg gtcagaagcg aaaacgagaacctgaagatg 793 agggagaaga tgatgactaa gtggaataac ctattttgaa aaattcctattgtgatttga 853 ctgtttttag ccgtatcccc tctccccccc cactctaatc ctgccccctg aa905 18 130 PRT Homo sapiens 18 Met Glu Met Gly Lys Trp Ile His Leu GluLeu Arg Asn Arg Thr Pro 1 5 10 15 Ser Asp Val Lys Glu Leu Phe Leu AspAsn Ser Gln Ser Asn Glu Gly 20 25 30 Lys Leu Glu Gly Leu Thr Asp Glu PheGlu Glu Leu Glu Leu Leu Asn 35 40 45 Thr Ile Asn Ile Gly Leu Thr Ser IleAla Asn Leu Pro Lys Leu Asn 50 55 60 Lys Leu Lys Lys Leu Glu Leu Ser SerAsn Arg Ala Ser Val Gly Leu 65 70 75 80 Glu Val Leu Ala Glu Lys Cys ProAsn Leu Ile His Leu Asn Leu Ser 85 90 95 Gly Asn Lys Ile Lys Asp Leu SerThr Ile Glu Pro Leu Lys Lys Leu 100 105 110 Glu Asn Leu Glu Ser Leu AspLeu Phe Thr Cys Glu Val Thr Asn Leu 115 120 125 Asn Asn 130 19 905 DNAHomo sapiens CDS (64)..(453) 19 gggttcgggg tttattggtt gaattccgctggctcaggag cctctgcaga gaaagcgtga 60 gag atg gag atg ggc aaa tgg att cattta gag ctg cgg aac agg acg 108 Met Glu Met Gly Lys Trp Ile His Leu GluLeu Arg Asn Arg Thr 1 5 10 15 ccc tcc gat gtg aaa gaa ctt ttc ctg gacaac agt cag tca aat gaa 156 Pro Ser Asp Val Lys Glu Leu Phe Leu Asp AsnSer Gln Ser Asn Glu 20 25 30 ggc aaa ttg gaa ggc ctc aca gat gaa ttt gaagaa ctg gaa tta tta 204 Gly Lys Leu Glu Gly Leu Thr Asp Glu Phe Glu GluLeu Glu Leu Leu 35 40 45 aat aca atc aac ata ggc ctc acc tca att gca aacttg cca aag tta 252 Asn Thr Ile Asn Ile Gly Leu Thr Ser Ile Ala Asn LeuPro Lys Leu 50 55 60 aac aaa ctt aag aag ctt gaa cta agc agt aac aga gcctca gtg ggc 300 Asn Lys Leu Lys Lys Leu Glu Leu Ser Ser Asn Arg Ala SerVal Gly 65 70 75 cta gaa gta ttg gca gaa aag tgt cca aac ctc ata cat ctaaat tta 348 Leu Glu Val Leu Ala Glu Lys Cys Pro Asn Leu Ile His Leu AsnLeu 80 85 90 95 agt ggc aac aaa att aaa gac ctc agc aca ata gag ccc ctgaaa aag 396 Ser Gly Asn Lys Ile Lys Asp Leu Ser Thr Ile Glu Pro Leu LysLys 100 105 110 tta gaa aac ctc gag agc tta gac ctt ttc act tgc gag gtaacc aac 444 Leu Glu Asn Leu Glu Ser Leu Asp Leu Phe Thr Cys Glu Val ThrAsn 115 120 125 ctg aac aac tactgagaaa agatgttcaa gctcctcctg caactcacat493 Leu Asn Asn 130 atctcaacgg ctgtgacccg gatgacaagg aggcccctaactcggatggt gagggctttg 553 tggagtgcct ggatgacaag gaggaggatg aggatgaggaggagtatgat gaagatgctc 613 aggtaatgga agatgaggag gacgaggatg aggaggaggaacgtgaagag gaggacgtga 673 gtggagacga ggaggagaag gatgaaggtt ataacaatggagaggtagat gatgaggaag 733 atgaagaaga gcttggtgaa gaagaaaggg gtcagaagcgaaaataagaa actgaagatg 793 agggagaaga cgatgcctaa gtggaataat ctattttgaaaaattccttt tgtgatttta 853 ctgtttttag ccgtatcccc tctccccccc cactctaatcctgccccctg aa 905 20 130 PRT Homo sapiens 20 Met Glu Met Gly Lys Trp IleHis Leu Glu Leu Arg Asn Arg Thr Pro 1 5 10 15 Ser Asp Val Lys Glu LeuPhe Leu Asp Asn Ser Gln Ser Asn Glu Gly 20 25 30 Lys Leu Glu Gly Leu ThrAsp Glu Phe Glu Glu Leu Glu Leu Leu Asn 35 40 45 Thr Ile Asn Ile Gly LeuThr Ser Ile Ala Asn Leu Pro Lys Leu Asn 50 55 60 Lys Leu Lys Lys Leu GluLeu Ser Ser Asn Arg Ala Ser Val Gly Leu 65 70 75 80 Glu Val Leu Ala GluLys Cys Pro Asn Leu Ile His Leu Asn Leu Ser 85 90 95 Gly Asn Lys Ile LysAsp Leu Ser Thr Ile Glu Pro Leu Lys Lys Leu 100 105 110 Glu Asn Leu GluSer Leu Asp Leu Phe Thr Cys Glu Val Thr Asn Leu 115 120 125 Asn Asn 13021 895 DNA Homo sapiens CDS (66)..(767) 21 gggttcgggg tttattgattgaattcggct ggcacgagag cctctgcaga cagagagcgc 60 gagag atg gag atg ggc agacgg att cat tca gag ctg cgg aac agg gcg 110 Met Glu Met Gly Arg Arg IleHis Ser Glu Leu Arg Asn Arg Ala 1 5 10 15 ccc tct gat gtg aaa gaa cttgtc ctg gac aac agt cgg tcg aat gaa 158 Pro Ser Asp Val Lys Glu Leu ValLeu Asp Asn Ser Arg Ser Asn Glu 20 25 30 ggc aaa ctc gaa gcc ctc aca gatgaa ttt gaa gaa ctg gaa ttc tta 206 Gly Lys Leu Glu Ala Leu Thr Asp GluPhe Glu Glu Leu Glu Phe Leu 35 40 45 agt aaa atc aac gga ggc ctc acc tcaatc tca gac tta cca aag tta 254 Ser Lys Ile Asn Gly Gly Leu Thr Ser IleSer Asp Leu Pro Lys Leu 50 55 60 aag ttg aga aag ctt gaa cta aaa gtc tcaggg ggc ctg gaa gta ttg 302 Lys Leu Arg Lys Leu Glu Leu Lys Val Ser GlyGly Leu Glu Val Leu 65 70 75 gca gaa aag tgt cca aac ctc acg cat cta tattta agt ggc aac aaa 350 Ala Glu Lys Cys Pro Asn Leu Thr His Leu Tyr LeuSer Gly Asn Lys 80 85 90 95 att aaa gac ctc agc aca ata gag cca ctg aaacag tta gaa aac ctc 398 Ile Lys Asp Leu Ser Thr Ile Glu Pro Leu Lys GlnLeu Glu Asn Leu 100 105 110 aag agc tta gac ctt ttc aat tgc gag gta accaac ctg aac gac tac 446 Lys Ser Leu Asp Leu Phe Asn Cys Glu Val Thr AsnLeu Asn Asp Tyr 115 120 125 gga gaa aac gtg ttc aag ctt ctc ctg caa ctcaca tat ctc gac agc 494 Gly Glu Asn Val Phe Lys Leu Leu Leu Gln Leu ThrTyr Leu Asp Ser 130 135 140 tgt tac tgg gac cac aag gag gcc cct tac tcagat att gag gac cac 542 Cys Tyr Trp Asp His Lys Glu Ala Pro Tyr Ser AspIle Glu Asp His 145 150 155 gtg gag ggc ctg gat gac gag gag gag ggt gagcat gag gag gag tat 590 Val Glu Gly Leu Asp Asp Glu Glu Glu Gly Glu HisGlu Glu Glu Tyr 160 165 170 175 gat gaa gat gct cag gta gtg gaa gat gaggag ggc gag gag gag gag 638 Asp Glu Asp Ala Gln Val Val Glu Asp Glu GluGly Glu Glu Glu Glu 180 185 190 gag gaa ggt gaa gag gag gac gtg agt ggaggg gac gag gag gat gaa 686 Glu Glu Gly Glu Glu Glu Asp Val Ser Gly GlyAsp Glu Glu Asp Glu 195 200 205 gaa ggt tat aac gat gga gag gta gat ggcgag gaa gat gaa gaa gag 734 Glu Gly Tyr Asn Asp Gly Glu Val Asp Gly GluGlu Asp Glu Glu Glu 210 215 220 ctt ggt gaa gaa gaa agg ggt cag aag cgaaaa tgagaacctg aagatgaggg 787 Leu Gly Glu Glu Glu Arg Gly Gln Lys ArgLys 225 230 agaagatgat gactaagtag aataacctat tttgaaaaat tcctattgtgatttgactgt 847 ttttacccat atcccccctc ccccccccct ctaatcctgc cccctgaa 89522 234 PRT Homo sapiens 22 Met Glu Met Gly Arg Arg Ile His Ser Glu LeuArg Asn Arg Ala Pro 1 5 10 15 Ser Asp Val Lys Glu Leu Val Leu Asp AsnSer Arg Ser Asn Glu Gly 20 25 30 Lys Leu Glu Ala Leu Thr Asp Glu Phe GluGlu Leu Glu Phe Leu Ser 35 40 45 Lys Ile Asn Gly Gly Leu Thr Ser Ile SerAsp Leu Pro Lys Leu Lys 50 55 60 Leu Arg Lys Leu Glu Leu Lys Val Ser GlyGly Leu Glu Val Leu Ala 65 70 75 80 Glu Lys Cys Pro Asn Leu Thr His LeuTyr Leu Ser Gly Asn Lys Ile 85 90 95 Lys Asp Leu Ser Thr Ile Glu Pro LeuLys Gln Leu Glu Asn Leu Lys 100 105 110 Ser Leu Asp Leu Phe Asn Cys GluVal Thr Asn Leu Asn Asp Tyr Gly 115 120 125 Glu Asn Val Phe Lys Leu LeuLeu Gln Leu Thr Tyr Leu Asp Ser Cys 130 135 140 Tyr Trp Asp His Lys GluAla Pro Tyr Ser Asp Ile Glu Asp His Val 145 150 155 160 Glu Gly Leu AspAsp Glu Glu Glu Gly Glu His Glu Glu Glu Tyr Asp 165 170 175 Glu Asp AlaGln Val Val Glu Asp Glu Glu Gly Glu Glu Glu Glu Glu 180 185 190 Glu GlyGlu Glu Glu Asp Val Ser Gly Gly Asp Glu Glu Asp Glu Glu 195 200 205 GlyTyr Asn Asp Gly Glu Val Asp Gly Glu Glu Asp Glu Glu Glu Leu 210 215 220Gly Glu Glu Glu Arg Gly Gln Lys Arg Lys 225 230 23 895 DNA Homo sapiensCDS (66)..(767) 23 gggttcgggg tttattgatt gaattccgcc ggcgcgggagcctctgcaga gagggagcgc 60 gagag atg gag atg ggc aga cgg att cat tta gagctg cgg aac agg acg 110 Met Glu Met Gly Arg Arg Ile His Leu Glu Leu ArgAsn Arg Thr 1 5 10 15 ccc tct gat gtg aaa gaa ctt gtc ctg gac aac agtcgg tcg aat gaa 158 Pro Ser Asp Val Lys Glu Leu Val Leu Asp Asn Ser ArgSer Asn Glu 20 25 30 ggc aaa ctc gaa ggc ctc aca gat gaa ttt gaa gaa ctggaa ttc tta 206 Gly Lys Leu Glu Gly Leu Thr Asp Glu Phe Glu Glu Leu GluPhe Leu 35 40 45 agt aca atc aac gta ggc ctc acc tca atc gca aac tta ccaaag tta 254 Ser Thr Ile Asn Val Gly Leu Thr Ser Ile Ala Asn Leu Pro LysLeu 50 55 60 aag ttg aga aag ctt gaa cta aga gtc tca ggg ggc ctg gaa gtattg 302 Lys Leu Arg Lys Leu Glu Leu Arg Val Ser Gly Gly Leu Glu Val Leu65 70 75 gca gaa aag tgt cca aac ctc acg cac cta tat tta agt ggc aac aaa350 Ala Glu Lys Cys Pro Asn Leu Thr His Leu Tyr Leu Ser Gly Asn Lys 8085 90 95 att aaa gac ctc agc aca ata gag cca ctg aaa cag tta gaa aac ctc398 Ile Lys Asp Leu Ser Thr Ile Glu Pro Leu Lys Gln Leu Glu Asn Leu 100105 110 aag agc tta gac ctt ttc aat tgc gag gta acc aac ctg aac gac tac446 Lys Ser Leu Asp Leu Phe Asn Cys Glu Val Thr Asn Leu Asn Asp Tyr 115120 125 gga gaa aac gtg ttc aag ctt ctc ctg caa ctc aca tat ctc gac agc494 Gly Glu Asn Val Phe Lys Leu Leu Leu Gln Leu Thr Tyr Leu Asp Ser 130135 140 tgt tac tgg gac cac aag gag gcc cct tac tca gat att gag gac cac542 Cys Tyr Trp Asp His Lys Glu Ala Pro Tyr Ser Asp Ile Glu Asp His 145150 155 gtg gag ggc ctg gat gac gag gag gag ggt gag cat gag gag gag tat590 Val Glu Gly Leu Asp Asp Glu Glu Glu Gly Glu His Glu Glu Glu Tyr 160165 170 175 gat gaa gat gct cag gta gtg gaa gat gag gag ggc gag gag ggggag 638 Asp Glu Asp Ala Gln Val Val Glu Asp Glu Glu Gly Glu Glu Gly Glu180 185 190 gag gaa ggt gaa gag gag gac gtg agt gga ggg gac gag gag gatgaa 686 Glu Glu Gly Glu Glu Glu Asp Val Ser Gly Gly Asp Glu Glu Asp Glu195 200 205 gaa ggt tat aac gat gga gag gta gat gac gag gaa gat gaa gaagag 734 Glu Gly Tyr Asn Asp Gly Glu Val Asp Asp Glu Glu Asp Glu Glu Glu210 215 220 ctt ggt gaa gaa gaa agg ggt cag aag cga aaa cgagaacctgaagatgaggg 787 Leu Gly Glu Glu Glu Arg Gly Gln Lys Arg Lys 225 230agaagatgat gactaagtgg aataacctat tttgaaaaat tcctattgtg atttgactgt 847ttttacccat atcccctctc ccccccccct ctaatcctgc cccctgaa 895 24 234 PRT Homosapiens 24 Met Glu Met Gly Arg Arg Ile His Leu Glu Leu Arg Asn Arg ThrPro 1 5 10 15 Ser Asp Val Lys Glu Leu Val Leu Asp Asn Ser Arg Ser AsnGlu Gly 20 25 30 Lys Leu Glu Gly Leu Thr Asp Glu Phe Glu Glu Leu Glu PheLeu Ser 35 40 45 Thr Ile Asn Val Gly Leu Thr Ser Ile Ala Asn Leu Pro LysLeu Lys 50 55 60 Leu Arg Lys Leu Glu Leu Arg Val Ser Gly Gly Leu Glu ValLeu Ala 65 70 75 80 Glu Lys Cys Pro Asn Leu Thr His Leu Tyr Leu Ser GlyAsn Lys Ile 85 90 95 Lys Asp Leu Ser Thr Ile Glu Pro Leu Lys Gln Leu GluAsn Leu Lys 100 105 110 Ser Leu Asp Leu Phe Asn Cys Glu Val Thr Asn LeuAsn Asp Tyr Gly 115 120 125 Glu Asn Val Phe Lys Leu Leu Leu Gln Leu ThrTyr Leu Asp Ser Cys 130 135 140 Tyr Trp Asp His Lys Glu Ala Pro Tyr SerAsp Ile Glu Asp His Val 145 150 155 160 Glu Gly Leu Asp Asp Glu Glu GluGly Glu His Glu Glu Glu Tyr Asp 165 170 175 Glu Asp Ala Gln Val Val GluAsp Glu Glu Gly Glu Glu Gly Glu Glu 180 185 190 Glu Gly Glu Glu Glu AspVal Ser Gly Gly Asp Glu Glu Asp Glu Glu 195 200 205 Gly Tyr Asn Asp GlyGlu Val Asp Asp Glu Glu Asp Glu Glu Glu Leu 210 215 220 Gly Glu Glu GluArg Gly Gln Lys Arg Lys 225 230 25 907 DNA Homo sapiens 25 gggttcggggtttattgatt gaattccgcc ggcgcgggag cctctgcaga gagagagcgc 60 gagagatggagatgggcaga cggattcatt tagagctgcg gaacaggacg ccctctgatg 120 tgaaagaacttgtcctggac aacagtcggt cgaatgaagg caaactcgag ggcctcacag 180 atgaatttgaagaactggaa ttcttaagta caatcaacgt aggcctcacc tcaatcgcaa 240 acttaccaaagttaaacaaa cttaagaagc ttgaactaag cgataacaga gtctcagggg 300 gcctggaagtattggcagaa aagtgtccga acctcacgca tctaaattta agtggcaaca 360 aaattaaagacctcagcaca atagagccac tgaaaaagtt agaaaacctc aagagcttag 420 accttttcaattgcgaggta accaacctga acgactaccg agaaaatgtg ttcaagctcc 480 tcccgcaactcacatatctc gacggctatg accgggacga caaggaggcc cctgactcgg 540 atgctgagggctacgtggag ggcctggatg atgaggagga ggatgaggat gaggaggagt 600 atgatgaagatgctcaggta gtggaagacg aggaggacga ggatgaggag gaggaaggtg 660 aagaggaggacgtgagtgga gaggaggagg aggatgaaga aggttataac gatggagagg 720 tagatgacgaggaagatgaa gaagagcttg gtgaagaaga aaggggtcag aagcgaaaac 780 gagaacctgaagatgaggga gaagatgatg actaagtgga ataacctatt ttgaaaaatt 840 cctattgtgatttgactgtt tttacccata tcccctctcc cccccccctc taatcctgcc 900 ccctgaa 90726 905 DNA Homo sapiens CDS (64)..(453) 26 gggttcgggg tttattggttgaattccgct ggctcaggag cctctgcaga gaaagcgtga 60 gag atg gag atg ggc aaatgg att cat tta gag ctg cgg aac agg acg 108 Met Glu Met Gly Lys Trp IleHis Leu Glu Leu Arg Asn Arg Thr 1 5 10 15 ccc tcc gat gtg aaa gaa cttttc ctg gac aac agt cag tca aat gaa 156 Pro Ser Asp Val Lys Glu Leu PheLeu Asp Asn Ser Gln Ser Asn Glu 20 25 30 ggc aaa ttg gaa ggc ctc aca gatgaa ttt gaa gaa ctg gaa tta tta 204 Gly Lys Leu Glu Gly Leu Thr Asp GluPhe Glu Glu Leu Glu Leu Leu 35 40 45 aat aca atc aac ata ggc ctc acc tcaatt gca aac ttg cca aag tta 252 Asn Thr Ile Asn Ile Gly Leu Thr Ser IleAla Asn Leu Pro Lys Leu 50 55 60 aac aaa ctt aag aag ctt gaa cta agc agtaac aga gcc tca gtg ggc 300 Asn Lys Leu Lys Lys Leu Glu Leu Ser Ser AsnArg Ala Ser Val Gly 65 70 75 cta gaa gta ttg gca gaa aag tgt cca aac ctcata cat cta aat tta 348 Leu Glu Val Leu Ala Glu Lys Cys Pro Asn Leu IleHis Leu Asn Leu 80 85 90 95 agt ggc aac aaa att aaa gac ctc agc aca atagag ccc ctg aaa aag 396 Ser Gly Asn Lys Ile Lys Asp Leu Ser Thr Ile GluPro Leu Lys Lys 100 105 110 tta gaa aac ctc gag agc tta gac ctt ttc acttgc gag gta acc aac 444 Leu Glu Asn Leu Glu Ser Leu Asp Leu Phe Thr CysGlu Val Thr Asn 115 120 125 ctg aac aac tactgagaaa agatgttcaa gctcctcctgcaactcacat 493 Leu Asn Asn 130 atctcaacgg ctgtgacccg gatgacaaggaggcccctaa ctcggatggt gagggctttg 553 tggagtgcct ggatgacaag gaggaggatgaggatgagga ggagtatgat gaagatgctc 613 aggtaatgga agatgaggag gacgaggatgaggaggagga acgtgaagag gaggacgtga 673 gtggagacga ggaggagaag gatgaaggttataacaatgg agaggtagat gatgaggaag 733 atgaagaaga gcttggtgaa gaagaaaggggtcagaagcg aaaataagaa actgaagatg 793 agggagaaga cgatgcctaa gtggaataatctattttgaa aaattccttt tgtgatttta 853 ctgtttttag ccgtatcccc tctccccccccactctaatc ctgccccctg aa 905 27 130 PRT Homo sapiens 27 Met Glu Met GlyLys Trp Ile His Leu Glu Leu Arg Asn Arg Thr Pro 1 5 10 15 Ser Asp ValLys Glu Leu Phe Leu Asp Asn Ser Gln Ser Asn Glu Gly 20 25 30 Lys Leu GluGly Leu Thr Asp Glu Phe Glu Glu Leu Glu Leu Leu Asn 35 40 45 Thr Ile AsnIle Gly Leu Thr Ser Ile Ala Asn Leu Pro Lys Leu Asn 50 55 60 Lys Leu LysLys Leu Glu Leu Ser Ser Asn Arg Ala Ser Val Gly Leu 65 70 75 80 Glu ValLeu Ala Glu Lys Cys Pro Asn Leu Ile His Leu Asn Leu Ser 85 90 95 Gly AsnLys Ile Lys Asp Leu Ser Thr Ile Glu Pro Leu Lys Lys Leu 100 105 110 GluAsn Leu Glu Ser Leu Asp Leu Phe Thr Cys Glu Val Thr Asn Leu 115 120 125Asn Asn 130 28 907 DNA Homo sapiens CDS (66)..(812) 28 gggttcggggtttattgatt gaattccgcc ggcgcgggag cctctgcaga gagagagcgc 60 gagag atg gagatg ggc aga cgg att cat cta gag ctg cgg aac agg acg 110 Met Glu Met GlyArg Arg Ile His Leu Glu Leu Arg Asn Arg Thr 1 5 10 15 ccc tct gat gtgaaa gaa ctt gtc ctg gtc aac agt cgg tcg aat gaa 158 Pro Ser Asp Val LysGlu Leu Val Leu Val Asn Ser Arg Ser Asn Glu 20 25 30 ggc aaa ctc gaa ggcctc aca gat gaa ttt gaa gaa ctg gaa ttc tta 206 Gly Lys Leu Glu Gly LeuThr Asp Glu Phe Glu Glu Leu Glu Phe Leu 35 40 45 agt aca atc aac gta ggcctc acc tca atc gca aac tta cca aag tta 254 Ser Thr Ile Asn Val Gly LeuThr Ser Ile Ala Asn Leu Pro Lys Leu 50 55 60 aac aaa ctt aag aag ctt gaacta agc gat aac aga gtc tca ggg ggc 302 Asn Lys Leu Lys Lys Leu Glu LeuSer Asp Asn Arg Val Ser Gly Gly 65 70 75 cta gaa gta ttg gca gaa aag tgtccg aac ctc acg cat cta aat tta 350 Leu Glu Val Leu Ala Glu Lys Cys ProAsn Leu Thr His Leu Asn Leu 80 85 90 95 agt ggc aac aaa att aaa gac ctcagc aca ata gag cca ctg aaa aag 398 Ser Gly Asn Lys Ile Lys Asp Leu SerThr Ile Glu Pro Leu Lys Lys 100 105 110 tta gaa aac ctc aag agc tta gacctt ttc aat tgc gag gta acc aac 446 Leu Glu Asn Leu Lys Ser Leu Asp LeuPhe Asn Cys Glu Val Thr Asn 115 120 125 ctg aac gac tac cga gaa aat gtgttc aag ctc ctc ccg caa ctc aca 494 Leu Asn Asp Tyr Arg Glu Asn Val PheLys Leu Leu Pro Gln Leu Thr 130 135 140 tat ctc gac ggc tat gac cgg gacgac aag gag gcc cct gac tcg gat 542 Tyr Leu Asp Gly Tyr Asp Arg Asp AspLys Glu Ala Pro Asp Ser Asp 145 150 155 gct gag ggc tac gtg gag ggc ctggat gat gag gag gag gat gag gat 590 Ala Glu Gly Tyr Val Glu Gly Leu AspAsp Glu Glu Glu Asp Glu Asp 160 165 170 175 gag gag gag tat gat gaa gatgct cag gta gtg gaa gac gag gag gac 638 Glu Glu Glu Tyr Asp Glu Asp AlaGln Val Val Glu Asp Glu Glu Asp 180 185 190 gag gat gag gag gag gaa ggtgaa gag gag gac gtg agt gga gag gag 686 Glu Asp Glu Glu Glu Glu Gly GluGlu Glu Asp Val Ser Gly Glu Glu 195 200 205 gag gag gat gaa gaa ggt tataac gat gga gag gta gat gac gag gaa 734 Glu Glu Asp Glu Glu Gly Tyr AsnAsp Gly Glu Val Asp Asp Glu Glu 210 215 220 gat gaa gaa gag ctt ggt gaagaa gaa agg ggt cag aag cga aaa cga 782 Asp Glu Glu Glu Leu Gly Glu GluGlu Arg Gly Gln Lys Arg Lys Arg 225 230 235 gaa cct gaa gat gag gga gaagat gat gac taagtggaat aacctatttt 832 Glu Pro Glu Asp Glu Gly Glu AspAsp Asp 240 245 gaaaaattcc tattgtgatt tgactgtttt tacccatatc ccctctcccccccccctcta 892 atcctgcccc ctgaa 907 29 249 PRT Homo sapiens 29 Met GluMet Gly Arg Arg Ile His Leu Glu Leu Arg Asn Arg Thr Pro 1 5 10 15 SerAsp Val Lys Glu Leu Val Leu Val Asn Ser Arg Ser Asn Glu Gly 20 25 30 LysLeu Glu Gly Leu Thr Asp Glu Phe Glu Glu Leu Glu Phe Leu Ser 35 40 45 ThrIle Asn Val Gly Leu Thr Ser Ile Ala Asn Leu Pro Lys Leu Asn 50 55 60 LysLeu Lys Lys Leu Glu Leu Ser Asp Asn Arg Val Ser Gly Gly Leu 65 70 75 80Glu Val Leu Ala Glu Lys Cys Pro Asn Leu Thr His Leu Asn Leu Ser 85 90 95Gly Asn Lys Ile Lys Asp Leu Ser Thr Ile Glu Pro Leu Lys Lys Leu 100 105110 Glu Asn Leu Lys Ser Leu Asp Leu Phe Asn Cys Glu Val Thr Asn Leu 115120 125 Asn Asp Tyr Arg Glu Asn Val Phe Lys Leu Leu Pro Gln Leu Thr Tyr130 135 140 Leu Asp Gly Tyr Asp Arg Asp Asp Lys Glu Ala Pro Asp Ser AspAla 145 150 155 160 Glu Gly Tyr Val Glu Gly Leu Asp Asp Glu Glu Glu AspGlu Asp Glu 165 170 175 Glu Glu Tyr Asp Glu Asp Ala Gln Val Val Glu AspGlu Glu Asp Glu 180 185 190 Asp Glu Glu Glu Glu Gly Glu Glu Glu Asp ValSer Gly Glu Glu Glu 195 200 205 Glu Asp Glu Glu Gly Tyr Asn Asp Gly GluVal Asp Asp Glu Glu Asp 210 215 220 Glu Glu Glu Leu Gly Glu Glu Glu ArgGly Gln Lys Arg Lys Arg Glu 225 230 235 240 Pro Glu Asp Glu Gly Glu AspAsp Asp 245 30 907 DNA Homo sapiens CDS (66)..(455) 30 gggttcggggtttattgatt gaattccgcc ggcgcgggag cctctgcaga gagagagcgc 60 gagag atg gagatg ggc aga cgg att cat tta gag ctg cgg aac agg acg 110 Met Glu Met GlyArg Arg Ile His Leu Glu Leu Arg Asn Arg Thr 1 5 10 15 ccc tct gat gtgaaa gaa ctt gtc ctg gac aac agt cgg tcg aat gaa 158 Pro Ser Asp Val LysGlu Leu Val Leu Asp Asn Ser Arg Ser Asn Glu 20 25 30 ggc aaa ctc gaa ggcctc aca gat gaa ttt gaa gaa ctg gaa ttc tta 206 Gly Lys Leu Glu Gly LeuThr Asp Glu Phe Glu Glu Leu Glu Phe Leu 35 40 45 agt aca atc aac gta ggcctc acc tca atc gca aac tta cca aag tta 254 Ser Thr Ile Asn Val Gly LeuThr Ser Ile Ala Asn Leu Pro Lys Leu 50 55 60 aac aaa ctt aag aag ctt gaacta agc gat aac aga gtc tca ggg ggc 302 Asn Lys Leu Lys Lys Leu Glu LeuSer Asp Asn Arg Val Ser Gly Gly 65 70 75 cta gaa gta ttg gca gaa aag tgtcca aac ctc ata cat cta aat tta 350 Leu Glu Val Leu Ala Glu Lys Cys ProAsn Leu Ile His Leu Asn Leu 80 85 90 95 agt ggc aac aaa att aaa gac ctcagc aca ata gag ccc ctg aaa aag 398 Ser Gly Asn Lys Ile Lys Asp Leu SerThr Ile Glu Pro Leu Lys Lys 100 105 110 tta gaa aac ctc gag agc tta gacctt ttc act tgc gag gta acc aac 446 Leu Glu Asn Leu Glu Ser Leu Asp LeuPhe Thr Cys Glu Val Thr Asn 115 120 125 ctg aac aac tactgagaaaagatgttcaa gctcctcctg caactcacat 495 Leu Asn Asn 130 atctcaacggctgtgacccg gatgacaagg aggcccctaa ctcggatggt gagggctttg 555 tggagtgcctggatgacaag gaggaggatg aggatgagga ggagtatgat gaagatgctc 615 aggtaatggaagatgaggag gacgaggatg aggaggagga acgtgaagag gaggacgtga 675 gtggagacgaggaggagaag gatgaaggtt ataacaatgg agaggtagat gatgaggaag 735 atgaagaagagcttggtgaa gaagaaaggg gtcagaagcg aaaataagaa actgaagatg 795 agggagaagacgatgcctaa gtggaataat ctattttgaa aaattcctat tgtgatttga 855 ctgtttttacccatatcccc tctccccccc ccctctaatc ctgccccctg aa 907 31 130 PRT Homosapiens 31 Met Glu Met Gly Arg Arg Ile His Leu Glu Leu Arg Asn Arg ThrPro 1 5 10 15 Ser Asp Val Lys Glu Leu Val Leu Asp Asn Ser Arg Ser AsnGlu Gly 20 25 30 Lys Leu Glu Gly Leu Thr Asp Glu Phe Glu Glu Leu Glu PheLeu Ser 35 40 45 Thr Ile Asn Val Gly Leu Thr Ser Ile Ala Asn Leu Pro LysLeu Asn 50 55 60 Lys Leu Lys Lys Leu Glu Leu Ser Asp Asn Arg Val Ser GlyGly Leu 65 70 75 80 Glu Val Leu Ala Glu Lys Cys Pro Asn Leu Ile His LeuAsn Leu Ser 85 90 95 Gly Asn Lys Ile Lys Asp Leu Ser Thr Ile Glu Pro LeuLys Lys Leu 100 105 110 Glu Asn Leu Glu Ser Leu Asp Leu Phe Thr Cys GluVal Thr Asn Leu 115 120 125 Asn Asn 130 32 908 DNA Homo sapiens 32gggttcgggg tttattgatt gaattccgcc ggcgcgggag cctctgcaga gagagagcgc 60ggagagatgg agatgggcag acggattcat ttagagctgc ggaacaggac gccctctgat 120gtgaaagaac ttgtcctgga caacagtcgg tcgaatgaag gcaaactcga aggcctcaca 180gatgaatttg aagaactgga attcttaagt acaatcaacg taggcctcac ctcaatcgca 240aacttaccaa agttaaacaa acttaagaag cttgaactaa gcgataacag agtctcaggg 300ggcctggaag tattggcaga aaagtgtccg aacctcacgc atctaaattt aagtggcaac 360aaaattaaag acctcagcac aatagagcca ctgaaaaagt tagaaaacct caagagctta 420gaccttttca attgcgaggt aaccaacctg aacgactacc gagaaaatgt gttcaagctc 480ctcccgcaac tcacatatct cgacggctat gaccgggacg acaaggaggc ccctgactcg 540gatgctgagg gctacgtgga gggcctggat gatgaggagg aggatgagga tgaggaggag 600tatgatgaag atgctcaggt agtggaagac gaggaggacg aggatgagga ggaggaaggt 660gaagaggagg acgtgagtgg agaggaggag gaggatgaag aaggttataa cgatggagag 720gtagatgacg aggaagatga agaagagctt ggtgaagaag aaaggggtca gaagcgaaaa 780cgagaacctg aagatgaggg agaagatgat gactaagtgg aataacctat tttgaaaaat 840tcctattgtg atttgactgt ttttacccat atcccctctc ccccccccct ctaatcctgc 900cccctgaa 908 33 906 DNA Homo sapiens CDS (66)..(812) 33 gggttcggggtttattgatt gaattccgct ggcgcgggag cctctgcaga gagagagcgc 60 gagag atg gagatg ggc aga cgg att cat tta gag ctg cgg aac agg acg 110 Met Glu Met GlyArg Arg Ile His Leu Glu Leu Arg Asn Arg Thr 1 5 10 15 ccc tct gat gtgaaa gaa ctt gtc ctg gac aac agt cgg tcg aat gaa 158 Pro Ser Asp Val LysGlu Leu Val Leu Asp Asn Ser Arg Ser Asn Glu 20 25 30 ggc aaa ctc gaa ggcctc aca gat gaa ttt gaa gaa ctg gaa ttc tta 206 Gly Lys Leu Glu Gly LeuThr Asp Glu Phe Glu Glu Leu Glu Phe Leu 35 40 45 agt aca atc aac gta ggcctc acc tca atc gca aac tta cca aag tta 254 Ser Thr Ile Asn Val Gly LeuThr Ser Ile Ala Asn Leu Pro Lys Leu 50 55 60 aac aaa ctt aag aag ctt gaacta agc agt aac aga gtc tca ggg ggc 302 Asn Lys Leu Lys Lys Leu Glu LeuSer Ser Asn Arg Val Ser Gly Gly 65 70 75 cta gaa gta ttg gca gaa aag tgtcca aac ctc acg cat cta aat tta 350 Leu Glu Val Leu Ala Glu Lys Cys ProAsn Leu Thr His Leu Asn Leu 80 85 90 95 agt ggc aac aaa att aaa gac ctcagc aca ata gag cca ctg aaa aag 398 Ser Gly Asn Lys Ile Lys Asp Leu SerThr Ile Glu Pro Leu Lys Lys 100 105 110 tta gaa aac ctc aag agc tta gacctt ttc aat tgc gag gta acc aac 446 Leu Glu Asn Leu Lys Ser Leu Asp LeuPhe Asn Cys Glu Val Thr Asn 115 120 125 ctg aac gac tac cga gaa aat gtgttc aag ctc ctc ctg caa ctc aca 494 Leu Asn Asp Tyr Arg Glu Asn Val PheLys Leu Leu Leu Gln Leu Thr 130 135 140 tat ctc gac ggc tgt gac cgg gacgac aag gag gcc cct gac tcg gat 542 Tyr Leu Asp Gly Cys Asp Arg Asp AspLys Glu Ala Pro Asp Ser Asp 145 150 155 gct gag ggc tac gtg gag ggc ctggat gac gag gag gag gat gag gat 590 Ala Glu Gly Tyr Val Glu Gly Leu AspAsp Glu Glu Glu Asp Glu Asp 160 165 170 175 gag gag gag tat gat gaa gatgct cag gta gtg gaa gat gag gag gac 638 Glu Glu Glu Tyr Asp Glu Asp AlaGln Val Val Glu Asp Glu Glu Asp 180 185 190 gag gat gag gag gag gaa ggtgaa gag gag gac gtg agt gga gag gag 686 Glu Asp Glu Glu Glu Glu Gly GluGlu Glu Asp Val Ser Gly Glu Glu 195 200 205 gag gag gat gaa gaa ggt tataac gat gga gag gta gat gac gag gaa 734 Glu Glu Asp Glu Glu Gly Tyr AsnAsp Gly Glu Val Asp Asp Glu Glu 210 215 220 gat gaa gaa gag ctt ggt gaagaa gaa agg ggt cag aag cga aaa gag 782 Asp Glu Glu Glu Leu Gly Glu GluGlu Arg Gly Gln Lys Arg Lys Glu 225 230 235 aac ctg aag atg agg gag aagatg atg act aagtggaata acctattttg 832 Asn Leu Lys Met Arg Glu Lys MetMet Thr 240 245 aaaaattcct attgtgattt gactgttttt acccatatcc cctctcccccccccctctaa 892 tcctgccccc tgaa 906 34 249 PRT Homo sapiens 34 Met GluMet Gly Arg Arg Ile His Leu Glu Leu Arg Asn Arg Thr Pro 1 5 10 15 SerAsp Val Lys Glu Leu Val Leu Asp Asn Ser Arg Ser Asn Glu Gly 20 25 30 LysLeu Glu Gly Leu Thr Asp Glu Phe Glu Glu Leu Glu Phe Leu Ser 35 40 45 ThrIle Asn Val Gly Leu Thr Ser Ile Ala Asn Leu Pro Lys Leu Asn 50 55 60 LysLeu Lys Lys Leu Glu Leu Ser Ser Asn Arg Val Ser Gly Gly Leu 65 70 75 80Glu Val Leu Ala Glu Lys Cys Pro Asn Leu Thr His Leu Asn Leu Ser 85 90 95Gly Asn Lys Ile Lys Asp Leu Ser Thr Ile Glu Pro Leu Lys Lys Leu 100 105110 Glu Asn Leu Lys Ser Leu Asp Leu Phe Asn Cys Glu Val Thr Asn Leu 115120 125 Asn Asp Tyr Arg Glu Asn Val Phe Lys Leu Leu Leu Gln Leu Thr Tyr130 135 140 Leu Asp Gly Cys Asp Arg Asp Asp Lys Glu Ala Pro Asp Ser AspAla 145 150 155 160 Glu Gly Tyr Val Glu Gly Leu Asp Asp Glu Glu Glu AspGlu Asp Glu 165 170 175 Glu Glu Tyr Asp Glu Asp Ala Gln Val Val Glu AspGlu Glu Asp Glu 180 185 190 Asp Glu Glu Glu Glu Gly Glu Glu Glu Asp ValSer Gly Glu Glu Glu 195 200 205 Glu Asp Glu Glu Gly Tyr Asn Asp Gly GluVal Asp Asp Glu Glu Asp 210 215 220 Glu Glu Glu Leu Gly Glu Glu Glu ArgGly Gln Lys Arg Lys Glu Asn 225 230 235 240 Leu Lys Met Arg Glu Lys MetMet Thr 245 35 26 DNA Homo sapiens 35 tatgctagcg ggttcggggt ttattg 26 3629 DNA Homo sapiens 36 gattctagat ggtaagtttg cgattgagg 29 37 29 DNA Homosapiens 37 gaatctagaa ggaggaggaa ggtgaagag 29 38 29 DNA Homo sapiens 38ctatctagat tcagggggca ggattagag 29 39 24 DNA Homo sapiens 39 gaggtttattgattgaattc ggct 24 40 24 DNA Homo sapiens 40 ccccagtaca cttttcccgt ctca24 41 12 DNA Artificial Sequence recognition sequence 41 tttttctttt tc12 42 10 DNA Artificial Sequence recognition sequence 42 ttaaaattca 1043 10 DNA Artificial Sequence recognition sequence 43 atgtaaaaca 10 4411 DNA Artificial Sequence recognition sequence 44 aagataaaac c 11 45 10DNA Artificial Sequence recognition sequence 45 ccactgggga 10 46 13 DNAArtificial Sequence recognition sequence 46 ctctctctct ctc 13 47 11 DNAArtificial Sequence recognition sequence 47 aaaacataaa t 11 48 131 PRTHomo sapiens 48 Met Glu Met Gly Lys Trp Ile His Leu Glu Leu Arg Asn ArgThr Pro 1 5 10 15 Ser Asp Val Lys Glu Leu Phe Leu Asp Asn Ser Gln SerAsn Glu Gly 20 25 30 Lys Leu Glu Gly Leu Ala Asp Glu Phe Glu Glu Leu GluLeu Leu Asn 35 40 45 Thr Ile Asn Ile Gly Leu Ser Ser Ile Ala Asn Leu AlaLys Leu Asn 50 55 60 Lys Leu Lys Lys Leu Glu Leu Ser Ser Asn Arg Ala SerVal Gly Leu 65 70 75 80 Glu Val Leu Ala Glu Lys Cys Pro Asn Leu Ile HisLeu Asn Leu Ser 85 90 95 Gly Asn Lys Ile Lys Asp Leu Ser Thr Ile Glu ProLeu Lys Lys Leu 100 105 110 Glu Asn Leu Glu Ser Leu Asp Leu Phe Thr CysGlu Val Thr Asn Leu 115 120 125 Asn Asn Tyr 130 49 234 PRT Homo sapiens49 Met Glu Met Gly Arg Arg Ile His Ser Glu Leu Arg Asn Arg Ala Pro 1 510 15 Ser Asp Val Lys Glu Leu Ala Leu Asp Asn Ser Arg Ser Asn Glu Gly 2025 30 Lys Leu Glu Ala Leu Thr Asp Glu Phe Glu Glu Leu Glu Phe Leu Ser 3540 45 Lys Ile Asn Gly Gly Leu Thr Ser Ile Ser Asp Leu Pro Lys Leu Lys 5055 60 Leu Arg Lys Leu Glu Leu Arg Val Ser Gly Gly Leu Glu Val Leu Ala 6570 75 80 Glu Lys Cys Pro Asn Leu Thr His Leu Tyr Leu Ser Gly Asn Lys Ile85 90 95 Lys Asp Leu Ser Thr Ile Glu Pro Leu Lys Gln Leu Glu Asn Leu Lys100 105 110 Ser Leu Asp Leu Phe Asn Cys Glu Val Thr Asn Leu Asn Asp TyrGly 115 120 125 Glu Asn Val Phe Lys Leu Leu Leu Gln Leu Thr Tyr Leu AspSer Cys 130 135 140 Tyr Trp Asp His Lys Glu Ala Pro Tyr Ser Asp Ile GluAsp His Val 145 150 155 160 Glu Gly Leu Asp Asp Glu Glu Glu Gly Glu HisGlu Glu Glu Tyr Asp 165 170 175 Glu Asp Ala Gln Val Val Glu Asp Glu GluGly Glu Glu Glu Glu Glu 180 185 190 Glu Gly Glu Glu Glu Asp Val Ser GlyGly Asp Glu Glu Asp Glu Glu 195 200 205 Gly Tyr Asn Asp Gly Glu Val AspGly Glu Glu Asp Glu Glu Glu Leu 210 215 220 Gly Glu Glu Glu Arg Gly GlnLys Arg Lys 225 230 50 17 DNA Homo sapiens 50 gggttcgggg tttattg 17 5120 DNA Homo sapiens 51 ctctaatcct gccccctgaa 20

1. An isolated DNA molecule comprising at least a sequence of 18contiguous nucleotides selected from the sequence consisting of basepairs 4894-4942 of the sequence in FIG. 2 or the corresponding sequencefrom FIG. 5, or a sequence complementary thereto, said DNA molecule alsocontaining non-mammalian DNA sequence and being substantially free ofhuman DNA molecules.
 2. An isolated DNA molecule comprising at least asequence of 18 contiguous nucleotides selected from a sequence whichencodes the amino acids from residue 146-163 of the amino acid sequenceof pp32r1 or the corresponding sequence of pp32r2.
 3. An isolatednucleic acid probe of at least 15 nucleotides which specificallyhybridizes on Northern blot with nucleic acid encoding the amino acidsfrom residue 146-163 of the amino acid sequence of pp32r1 or thecorresponding sequence of pp32r2.
 4. An isolated nucleic acid probecomprising a sequence of at least 8 contiguous nucleotides unique topp32r1 or pp32r2.
 5. A nucleic acid molecule produced by recombinantmethods, wherein said nucleic acid molecule encodes at least the aminoacids from residue 146-163 of sequence of the amino acid sequence ofpp32r1 or the corresponding sequence of pp32r2.
 6. The nucleic acidmolecule according to claim 5, wherein said nucleic acid molecule is anexpression vector which expresses said amino acid sequence.
 7. Arecombinant cell containing the nucleic acid molecule of claim
 6. 8. Anucleic acid molecule produced by recombinant methods, said nucleic acidmolecule containing a sequence encoding at least the amino acids fromresidue 146-163 of sequence of the amino acid sequence of pp32r1 or thecorresponding sequence of pp32r2, said sequence being operatively linkedto a promoter in antisense orientation.
 9. A pair of nucleic acidprimers each of which comprises at least 10 contiguous nucleotides, atleast one of said primers being selected from or complementary to thesequence of pp32r1, wherein nucleic acid amplification of humanchromosome 4 or a transcript thereof using said pair of nucleic acidprimers will produce an amplified nucleic acid encoding residues 146-163of the sequence of pp32r1.
 10. A diagnostic method for predictingmalignant potential of neuroendocrine, neural, mesenchymal, lymphoid,epithelial or germ cell derived tumors, comprising: providing a sampleof human neuroendocrine, neural, mesenchymal, lymphoid, epithelial orgerm cell derived tissue; and determining, in the sample, levels orintracellular sites of expression of a gene product expressed from agene sequence which encodes residues 146-163 of the sequence of pp32r1or the corresponding sequence of pp32r2.
 11. A diagnostic method forpredicting malignant potential of neuroendocrine, neural, mesenchymal,lymphoid, epithelial or germ cell derived tumors, comprising: providinga sample of human neuroendocrine, neural, mesenchymal, lymphoid,epithelial or germ cell derived tumor tissue; and determining, in thesample, levels or intracellular sites of expression of a gene productexpressed from a gene sequence which encodes residues 146-163 of thesequence of pp32r1 or the corresponding sequence of pp32r2.
 12. Themethod of claim 11, wherein the gene product is mRNA.
 13. The method ofclaim 12, wherein the mRNA is extracted from the sample and quantitated.14. The method of claim 12, wherein the level of mRNA is determined byin situ hybridization to a section of the tissue sample.
 15. The methodof claim 12, wherein the mRNA is quantitated by polymerase chainreaction.
 16. The method according to claim 11, wherein the gene productis protein.
 17. The method according to claim 16, wherein the methodfurther comprises reacting the sample with an antibody that specificallybinds to a polypeptide consisting of the sequence of pp32r1, but doesnot specifically bind to a polypeptide consisting of the sequence ofpp32 or pp32r2, or an antibody that specifically binds to a polypeptideconsisting of the sequence of pp32r2, but does not specifically bind toa polypeptide consisting of the sequence of pp32 or pp32r1
 18. Themethod according to claim 11, wherein the tissue is a carcinoma.
 19. Themethod according to claim 11, wherein the tissue is a carcinoma orsarcoma of a tissue selected from the group consisting of epithelial,lymphoid, hematopoietic, mesenchymal, central nervous system andperipheral nervous system tissues.
 20. The method according to claim 19,wherein the tissue is selected from the group consisting of coloncarcinoma, prostate carcinoma and non-Hodgkin's lymphoma.
 21. Anantibody that specifically binds to a polypeptide consisting of thesequence of pp32r1 or pp32r2, but does not specifically bind to apolypeptide consisting of the sequence of pp32.
 22. The antibody ofclaim 21, wherein the antibody is a monoclonal antibody.
 23. An isolatedDNA molecule comprising an androgen-activated transcriptional promoter.24. The isolated DNA molecule of claim 23, wherein the promotercomprises a transcription initiation site and a binding site for asteroid hormone receptor protein positioned within 10,000 nucleotidebase pairs (bp) of the transcription initiation site, preferably 5,000bp, more preferably 3000 bp.
 25. The isolated DNA molecule of claim 24,further comprising at least one binding site for steroid hormonereceptor proteins positioned within 2000 nucleotide base pairs (bp) ofthe transcription initiation site, preferably a plurality of bindingsites for steroid hormone receptor proteins are positioned within 2000bp of the transcription initiation site, more preferably, at least 5binding sites for steroid hormone receptor proteins are so positioned.26. The isolated DNA molecule of claim 25, wherein the binding sites forsteroid hormone receptor proteins are selected from the group of steroidreceptor protein binding sites listed on Table
 1. 27. The isolated DNAmolecule of claim 24, further comprising an open reading framecomprising at least one exon of a protein coding sequence, wherein saidopen reading frame is operatively linked to said androgen-activatedtranscriptional promoter.
 28. The isolated DNA molecule of claim 27,wherein transcriptional activity of the promoter is regulated bysteroids.
 29. A method of screening a compound for pharmacologicalactivity comprising: culturing a cell transfected with the DNA moleculeof claim 27; and determining expression of the protein coding sequencein the presence and absence of the compound.
 30. The method of claim 29,wherein the expression determined is RNA expression or proteinexpression.
 31. The DNA molecule of claim 23, wherein the DNA moleculeis a DNA vector.